Biosynthesis of commodity chemicals

ABSTRACT

Methods, enzymes, recombinant microorganism, and microbial systems are provided for converting suitable monosaccharides or oligosaccharides, such as those derived from biomass, as well as various aldehydes and/or ketones, into commodity chemicals, such as biofuels. Commodity chemicals produced by the methods described herein are also provided. Commodity chemical enriched, refinery-produced petroleum products are also provided, as well as methods for producing the same.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of copending U.S. patent application Ser. No. 12/636,046, with a filing date of Dec. 11, 2009, which claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61/121,869 filed Dec. 11, 2008, all of which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 690212000901SEQLIST.txt, date recorded: Jul. 18, 2011, size: 179 KB).

TECHNICAL FIELD

The present application relates generally to the use of recombinant microorganisms and chemical/enzymatic systems to convert aldehydes and/or ketones to various commodity chemicals or biofuels, such as isooctane.

BACKGROUND

Petroleum is facing declining global reserves and contributes to more than 30% of greenhouse gas emissions driving global warming. Annually 800 billion barrels of transportation fuel are consumed globally. Diesel and jet fuels account for greater than 50% of global transportation fuels.

Significant legislation has been passed, requiring fuel producers to cap or reduce the carbon emissions from the production and use of transportation fuels. Fuel producers are seeking substantially similar, low carbon fuels that can be blended and distributed through existing infrastructure (e.g., refineries, pipelines, tankers).

Due to increasing petroleum costs and reliance on petrochemical feedstocks, the chemical industry is also looking for ways to improve margin and price stability, while reducing its environmental footprint. The chemical industry is striving to develop greener products that are more energy, water, and CO₂ efficient than current products. Fuels produced from biological sources, such as biomass, represent one aspect of that process.

Many present methods for converting biomass into biofuels focus on the use of lignocellulolic biomass. However, there are many problems associated with using this process. Large-scale cultivation of lignocellulolic biomass requires a substantial amount of cultivated land, which can be only achieved by replacing food crop production with energy crop production, deforestation, and by recultivating currently uncultivated land. Other problems include a decrease in water availability and quality as well as an increase in the use of pesticides and fertilizers.

The degradation of lignocellulolic biomass using biological systems presents a significant challenge due to its substantial mechanical strength and the complex chemical components. Approximately thirty different enzymes are required to fully convert lignocellulose to monosaccharides. The only available alternate to this complex approach requires a substantial amount of heat, pressure, and strong acids. The art therefore needs an economic and technically simple process for converting biomass into hydrocarbons for use as biofuels or biopetrols. U.S. application Ser. Nos. 12/245,537 and 12/245,540 describe the use of recombinant microorganisms to produce various biofuels from biomass, and also describe the use of such recombinant microorganisms to produce various aldehydes, such as butyraldehyde and isobutyraldehyde, from biomass derived saccharides.

2,2,4-Trimethylpentane, also known as isooctane, is an octane isomer that defines the 100 point on the octane rating scale. Isooctane represents an important component of gasoline. Isooctane is produced on a massive scale in the petroleum industry, often as a mixture with related hydrocarbons. The petroleum industry typically relies on the alkylation process to produce isooctane, which alkylates isobutane with isobutylene using a strong acid catalyst.

Moreover, existing petroleum reserves are less and less useful for gasoline because of low octane content, and the ability to add octane can increase the useability of current petroleum reserves. The art therefore needs an environmentally friendly and technically simple process for producing isooctane, as well as other related biofuels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary pathway by which isobutyraldehyde may be converted in vivo to 2,4,4-trimethyl-1,3-pentanediol.

FIG. 2 shows the production of 3-hydroxy-2,2,4-trimethylpentanal from DH10B strain harboring pTrcTM1559 (A) and its control plasmid (B), as measured by GC-MS.

FIG. 3 shows the production of 2-ethyl-2-hexene-1-al from DH10B strain harboring pTrcTM1559 (A) and its control plasmid (B), as measured by GC-MS.

FIG. 4 shows the production of 2-butyl-2-octene-1-al from DH10B strain harboring pTrcTM1559 (A) and its control plasmid (B), as measured by GC-MS

BRIEF SUMMARY

Embodiments of the present invention relate generally to methods of producing a commodity chemical, or an intermediate thereof, comprising growing a recombinant microorganism with a source of an aldehyde, a ketone, or both, wherein the recombinant microorganism comprises: (i) at least one exogenous polynucleotide encoding and expressing a polypeptide having an aldolase activity; and (ii) at least one exogenous polynucleotide encoding and expressing a polypeptide having an alcohol dehydrogenase activity, wherein at least one of the polynucleotides is exogenous, thereby producing the commodity chemical, or the intermediate thereof.

In certain embodiments, the commodity chemical, or the intermediate thereof, is selected from the following formulas:

wherein R1 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, and CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂; and

wherein R2 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂,

wherein n=0-30. In certain embodiments, the commodity chemical is further enzymatically or chemically converted to its corresponding alkane.

In certain embodiments, the commodity chemical is selected from the group consisting of 3-hydroxy-2,2,4 trimethylpentanal and 2,2,4-trimethyl-1,3-pentanediol. In certain embodiments, the 2,2,4-trimethyl-1,3-pentanediol is further enzymatically or chemically converted to 2,2,4-trimethylpentane.

In certain embodiments, the commodity chemical is selected from the group consisting of 3-hydroxy-2-ethylhexanal, 2-ethyl-2-hexene-1-al, 2-ethylhexanal, and 2-ethylhexanol. In certain embodiments, the 2-ethylhexanol is further enzymatically or chemically converted to 2-ethylhexane.

In certain embodiments, the commodity chemical is selected from the group consisting of 3-hydroxy-2-butyl-1-octanol, 2-butyl-2-octene-1-al, 2-butyl-octanal, and 2-butyl-octanol. In certain embodiments, the 2-butyl-octanol is further enzymatically or chemically converted to 2-butyl-octane.

In certain embodiments, the source of the aldehyde, ketone, or both is a recombinant microorganism that comprises an aldehyde and/or ketone biosynthesis pathway selected from an acetoaldehyde, propionaldehyde, glutaraldehyde, butyraldehyde, isobutyraldehyde, 2-methyl-butyraldehyde, 3-methyl-butyraldehyde, 4-methylpentaldehyde, hexanaldehyde, octanaldehyde, phenylacetoaldehyde, 2-phenyl acetoaldehyde, 2-(4-hydroxyphenyl)acetoaldehyde, 2-Indole-3-acetoaldehyde, 5-amino-pentaldehyde, succinate semialdehyde, and a succinate 4-hydroxyphenyl acetaldehyde biosynthesis pathway, and combinations thereof.

In certain embodiments, the recombinant microorganism that comprises the aldehyde and/or ketone biosynthesis pathway is the same as the recombinant microorganism that produces or synthesizes the commodity chemical. In certain embodiments, the recombinant microorganism that comprises the aldehyde and/or ketone biosynthesis pathway is different than the recombinant microorganism that produces or synthesizes the commodity chemical.

In certain embodiments, the aldehyde and/or ketone biosynthesis pathway comprises an isobutyraldehyde biosynthesis pathway, and wherein the aldehyde is isobutyraldehyde. In certain embodiments, the aldehyde and/or ketone biosynthesis pathway comprises a butyraldehyde biosynthesis pathway, and wherein the aldehyde is butyraldehyde. In certain embodiments, the aldehyde and/or ketone biosynthesis pathway comprises a hexanaldehyde biosynthesis pathway, and wherein the aldehyde is hexanaldehyde.

In certain embodiments, the at least one exogenous polynucleotide encoding and expressing the polypeptide having the aldolase activity comprises (i) a nucleotide sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to at least one nucleotide sequence set forth in SEQ ID NOS:51-82, or (ii) a nucleotide sequence that hybridizes to at least one nucleotide sequence set forth in SEQ ID NOS:51-82, or a complement thereof, under conditions of medium stringency. In certain embodiments, the polypeptide having the aldolase activity comprises at least one biologically active motif selected from the amino acid sequences set forth in SEQ ID NOS:223-244, 255-260.

In certain embodiments, the at least one exogenous polynucleotide encoding and expressing the polypeptide having the alcohol dehydrogenase activity comprises (i) a nucleotide sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 30, 31, 33, or 83-96, or (ii) a nucleotide sequence that hybridizes to at least one nucleotide sequence set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 30, 31, 33, or 83-96, or a complement thereof, under conditions of medium stringency. In certain embodiments, the polypeptide having the alcohol dehydrogenase activity comprises at least one of a nicotinamide adenine dinucleotide (NAD+), a NADH, nicotinamide adenine dinucleotide phosphate (NADP+), or a NADPH binding motif. In certain embodiments, the NAD+, NADH, NADP+, or NADPH binding motif is selected from the group consisting of Y-X-G-G-X-Y (SEQ ID NO:245), Y-X-X-G-G-X-Y (SEQ ID NO:246), Y-X X-X-G-G-X-Y (SEQ ID NO:247), Y-X-G-X-X-Y (SEQ ID NO:248), Y-X-X-G-G-X-X-Y (SEQ ID NO:249), Y-X-X-X-G-X-X-Y (SEQ ID NO:250), Y-X-G-X-Y (SEQ ID NO:251), Y-X-X-G-X-Y (SEQ ID NO:252), Y-X-X-X-G-X-Y (SEQ ID NO:253), and Y-X-X-X-X-G-X-Y (SEQ ID NO:254); wherein Y is independently selected from alanine, glycine, and serine, wherein G is glycine, and wherein X is independently selected from a genetically encoded amino acid.

In certain embodiments, the recombinant microorganism further comprises at least one exogenous polynucleotide encoding and expressing a polypeptide having a double bond reductase activity and/or at least one polynucleotide encoding and expressing a polypeptide having a dehydratase activity. In certain embodiments, the at least one exogenous polynucleotide encoding and expressing the polypeptide having the double bond reductase activity comprises (i) a nucleotide sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NOS:35-50, or (ii) a nucleotide sequence that hybridizes to at least one nucleotide sequence set forth in SEQ ID NOS:35-50, or a complement thereof, under conditions of medium stringency.

Embodiments of the present invention also relate generally to recombinant microorganisms, comprising (i) at least one exogenous polynucleotide encoding and expressing a polypeptide having an aldolase activity; and (ii) at least one exogenous polynucleotide encoding and expressing a polypeptide having an alcohol dehydrogenase activity.

In certain embodiments, the recombinant microorganism is capable of converting a source of an aldehyde, a ketone, or both, to a commodity chemical, or an intermediate thereof, wherein the commodity chemical, or the intermediate thereof, is selected from the following formulas:

wherein R1 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, and CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂; and

wherein R2 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂,

wherein n=0-30.

In certain embodiments, the recombinant microorganism further comprises (iii) at least one exogenous polynucleotide encoding and expressing an aldehyde and/or ketone biosynthesis pathway. In certain embodiments, the microorganism that further comprises the aldehyde and/or ketone biosynthesis pathway is capable of converting a suitable monosaccharide or suitable oligosaccharide to a commodity chemical, or an intermediate thereof, wherein the commodity chemical, or the intermediate thereof, is selected from the following formulas:

wherein R1 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, and CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂; and

wherein R2 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂,

wherein n=0-30.

In certain embodiments, the at least one exogenous polynucleotide encoding and expressing the aldehyde and/or ketone biosynthesis pathway comprises a pathway selected from an acetoaldehyde, propionaldehyde, glutaraldehyde, butyraldehyde, isobutyraldehyde, 2-methyl-butyraldehyde, 3-methyl-butyraldehyde, 4-methylpentaldehyde, hexanaldehyde, octanaldehyde, phenylacetoaldehyde, 2-phenyl acetoaldehyde, 2-(4-hydroxyphenyl)acetoaldehyde, 2-Indole-3-acetoaldehyde, 5-amino-pentaldehyde, succinate semialdehyde, and succinate 4-hydroxyphenyl acetaldehyde biosynthesis pathway, and combinations thereof.

In certain embodiments, the aldehyde and/or ketone biosynthesis pathway comprises an isobutyraldehyde biosynthesis pathway, and wherein the aldehyde is isobutyraldehyde. In certain embodiments, the aldehyde and/or ketone biosynthesis pathway comprises a butyraldehyde biosynthesis pathway, and wherein the aldehyde is butyraldehyde. In certain embodiments, the aldehyde and/or ketone biosynthesis pathway comprises a hexanaldehyde biosynthesis pathway, and wherein the aldehyde is hexanaldehyde.

In certain embodiments, the at least one exogenous polynucleotide encoding and expressing the polypeptide having the aldolase activity comprises (i) a nucleotide sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to at least one nucleotide sequence set forth in SEQ ID NOS:51-82, or (ii) a nucleotide sequence that hybridizes to at least one nucleotide sequence set forth in SEQ ID NOS:51-82, or a complement thereof, under conditions of medium stringency. In certain embodiments, the polypeptide having the aldolase activity comprises at least one biologically active motif selected from the amino acid sequences set forth in SEQ ID NOS:223-244, 255-260.

In certain embodiments, the at least one exogenous polynucleotide encoding and expressing the polypeptide having the alcohol dehydrogenase activity comprises (i) a nucleotide sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 30, 31, 33, or 83-96, or (ii) a nucleotide sequence that hybridizes to at least one nucleotide sequence set forth in SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 30, 31, 33, or 83-96, or a complement thereof, under conditions of medium stringency. In certain embodiments, the polypeptide having the alcohol dehydrogenase activity comprises at least one of a nicotinamide adenine dinucleotide

(NAD+), a NADH, nicotinamide adenine dinucleotide phosphate (NADP+), or a NADPH binding motif. In certain embodiments, the NAD+, NADH, NADP+, or NADPH binding motif is selected from the group consisting of Y-X-G-G-X-Y (SEQ ID NO:245), Y-X-X-G-G-X-Y (SEQ ID NO:246), Y-X-X-X-G-G-X-Y (SEQ ID NO:247), Y-X-G-X-X-Y (SEQ ID NO:248), Y-X-X-G-G-X-X-Y (SEQ ID NO:249), Y-X-X-X-G-X-X-Y (SEQ ID NO:250), Y-X-G-X-Y (SEQ ID NO:251), Y-X-X-G-X-Y (SEQ ID NO:252), Y-X-X-X-G-X-Y (SEQ ID NO:253), and Y-X-X-X-X-G-X-Y (SEQ ID NO:254); wherein Y is independently selected from alanine, glycine, and serine, wherein G is glycine, and wherein X is independently selected from a genetically encoded amino acid.

In certain embodiments, the recombinant microorganism further comprises at least one exogenous polynucleotide encoding and expressing a polypeptide having a double bond reductase activity and/or at least one polynucleotide encoding and expressing a polypeptide having a dehydratase activity. In certain embodiments, the at least one exogenous polynucleotide encoding and expressing the polypeptide having the double bond reductase activity comprises (i) a nucleotide sequence that is at least 80%, 90%, 95%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NOS:35-50, or (ii) a nucleotide sequence that hybridizes to at least one nucleotide sequence set forth in SEQ ID NOS:35-50, or a complement thereof, under conditions of medium stringency.

Embodiments of the present invention also relate generally to methods of producing a commodity chemical, or an intermediate thereof, comprising growing a first recombinant microorganism with a source of a suitable monosaccharide or oligosaccharide, wherein the first recombinant microorganism comprises, (i) an aldehyde or ketone biosynthesis pathway, (ii) at least one polynucleotide encoding and expressing a polypeptide having an aldolase activity; and (iii) at least one polynucleotide encoding and expressing a polypeptide having an alcohol dehydrogenase activity, wherein at least one of the polynucleotides is exogenous, thereby producing the commodity chemical, or the intermediate thereof. In certain embodiments, the source of the suitable monosaccharide or oligosaccharide comprises a second recombinant microorganism that is capable of growing on a biomass-derived polysaccharide as a sole source of carbon. In certain embodiments, the biomass-derived polysaccharide is selected from alginate and pectin.

Embodiments of the present invention also relate generally to methods of producing a commodity chemical, or an intermediate thereof, comprising growing a recombinant microorganism with a biomass-derived polysaccharide, wherein the recombinant microorganism is capable of growing on the biomass-derived polysaccharide as a sole source of carbon, and wherein the recombinant microorganism comprises (i) an aldehyde or ketone biosynthesis pathway, (ii) at least one polynucleotide encoding and expressing a polypeptide having an aldolase activity; and (iii) at least one polynucleotide encoding and expressing a polypeptide having an alcohol dehydrogenase activity, wherein at least one of the polynucleotides is exogenous, thereby producing the commodity chemical, or the intermediate thereof. In certain embodiments, the biomass-derived polysaccharide is selected from alginate and pectin.

Embodiments of the present invention also relate to recombinant microorganisms, comprising (i) an aldehyde or ketone biosynthesis pathway, (ii) at least one exogenous polynucleotide encoding and expressing a polypeptide having an aldolase activity; and (iii) at least one exogenous polynucleotide encoding and expressing a polypeptide having an alcohol dehydrogenase activity. In certain embodiments, the recombinant microorganism is capable of growing on a biomass-derived polysaccharide as a sole source of carbon.

In certain embodiments, the microorganism is selected from Acetobacter aceti, Achromobacter, Acidiphilium, Acinetobacter, Actinomadura, Actinoplanes, Aeropyrum pernix, Agrobacterium, Alcaligenes, Ananas comosus (M), Arthrobacter, Aspargillus niger, Aspargillus oryze, Aspergillus melleus, Aspergillus pulverulentus, Aspergillus saitoi, Aspergillus sojea, Aspergillus usamii, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus lentus, Bacillus licheniformis, Bacillus macerans, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium, Brevibacillus brevis, Burkholderia cepacia, Candida cylindracea, Candida rugosa, Carica papaya (L), Cellulosimicrobium, Cephalosporium, Chaetomium erraticum, Chaetomium gracile, Clostridium, Clostridium butyricum, Clostridium acetobutylicum, Clostridium thermocellum, Corynebacterium (glutamicum), Corynebacterium efficiens, Escherichia coli, Enterococcus, Erwina chrysanthemi, Gliconobacter, Gluconacetobacter, Haloarcula, Humicola insolens, Humicola nsolens, Kitasatospora setae, Klebsiella, Klebsiella oxytoca, Kluyveromyces, Kluyveromyces fragilis, Kluyveromyces lactis, Kocuria, Lactlactis, Lactobacillus, Lactobacillus fermentum, Lactobacillus sake, Lactococcus, Lactococcus lactis, Leuconostoc, Methylocystis, Methanolobus siciliae, Methanogenium organophilum, Methanobacterium bryantii, Microbacterium imperiale, Micrococcus lysodeikticus, Microlunatus, Mucor javanicus, Mycobacterium, Myrothecium, Nitrobacter, Nitrosomonas, Nocardia, Papaya carica, Pediococcus, Pediococcus halophilus, Penicillium, Penicillium camemberti, Penicillium citrinum, Penicillium emersonii, Penicillium roqueforti, Penicillum lilactinum, Penicillum multicolor, Paracoccus pantotrophus, Propionibacterium, Pseudomonas, Pseudomonas fluorescens, Pseudomonas denitrificans, Pyrococcus, Pyrococcus furiosus, Pyrococcus horikoshii, Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Rhizopus oryzae, Rhizopus oligosporus, Rhodococcus, Sccharomyces cerevisiae, Sclerotina libertina, Sphingobacterium multivorum, Sphingobium, Sphingomonas, Streptococcus, Streptococcus thermophilus Y-1, Streptomyces, Streptomyces griseus, Streptomyces lividans, Streptomyces murinus, Streptomyces rubiginosus, Streptomyces violaceoruber, Streptoverticillium mobaraense, Tetragenococcus, Thermus, Thiosphaera pantotropha, Trametes, Trichoderma, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon penicillatum, Vibrio alginolyticus, Xanthomonas, yeast, Zygosaccharomyces rouxii, Zymomonas, and Zymomonus mobilis.

Embodiments of the present invention also relate to commodity chemicals produced by any of the methods or recombinant microorganisms described herein. Certain embodiments also include blended commodity chemicals comprising any of the commodity chemicals described herein and a refinery-produced petroleum product. In certain embodiments, the commodity chemical is selected from isooctane, 2-ethylhexane, and 2-butyl-octane. In certain embodiments, the refinery-produced petroleum product is selected from gasoline, jet fuel, and diesel fuel.

The present invention also relates to methods of producing a commodity chemical enriched refinery-produced petroleum product, comprising (a) blending a refinery-produced petroleum product with any of the commodity chemicals produced by the methods or recombinant microorganisms described herein, thereby producing the commodity chemical enriched refinery-produced petroleum product.

DETAILED DESCRIPTION

Embodiments of the present invention relate to the discovery that recombinant microorganisms can be engineered to utilize various aldehydes and/or ketones in the production of a variety of commodity chemicals or biofuels. For instance, the insertion of one or more exogenous polynucleotides that encode an enzyme having an aldolase activity and one or more exogenous polynucleotides that encode an enzyme having an alcohol dehydrogenase activity may render a microorganism capable of converting aldehydes and/or ketones into various commodity chemicals, or intermediates thereof. In certain aspects, these commodity chemicals may then be further chemically or enzymatically converted to other commodity chemicals, including biofuels. Such biofuels may include, for example, medium to long chain alkanes, such as isooctane and 2-ethylhexane.

In certain embodiments, the methods and recombinant microorganisms provided herein may be utilized to produce a variety of commodity chemicals, especially medium to long chain hydrocarbons. For instance, recombinant microorganisms of the present invention may be utilized generally to catalyze the following reactions:

wherein R1 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, and CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂; and

wherein R2 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂,

wherein n=0-30, and any corresponding alkanes produced therefrom.

Typically, the first chemical from left to right in the reaction exemplified above is produced from the condensation of two aldehydes and/or ketones, which may be the same or different, and which may be catalyzed by an enzyme having an aldolase activity, as described herein and known in the art.

After the aldolase condensation step, the first step in the reaction exemplified above typically occurs spontaneously, or may be catalyzed by a dehydratase. The second reaction may be catalyzed by an endogenous double bond reductase, or may be enhanced by the addition of an exogenous double bond reductase. The third reaction to produce the diol or alcohol may be catalyzed by an alcohol dehydrogenase, as described herein and known in the art. Thus, certain recombinant microorganisms of the invention may comprise an exogenous aldolase, optionally an exogenous double bond reductase, and/or an exogenous alcohol dehydrogenase.

As noted above, 2,2,4-trimethylpentane, also known as isooctane, is an octane isomer that defines the 100 point on the octane rating scale, and represents an important component of gasoline. As one particular illustration of the in vivo biological production of this molecule, a biosynthetic pathway to produce isooctane may be initiated in a recombinant microorganism that has access to a source of isobutyraldehyde. In certain embodiments, isobutyraldehyde may be obtained from a recombinant microorganism that comprises a isobutyraldehyde biosynthesis pathway, as described herein, and which can convert a suitable monosaccharide or oligosaccharide to isobutyraldehyde.

Briefly, regardless of the source of isobutyraldehyde, a recombinant microorganism of the invention may be utilized to condense two molecules of isobutyraldehyde to form 3-hydroxy-2,2,4-trimethyl pentanal, which has been shown herein to be catalyzed in vivo by a recombinant microorganism that comprises an exogenous enzyme having an aldolase activity. In certain embodiments, 3-hydroxy-2,2,4-trimethyl pentanal may then be readily reduced in vivo to 2,2,4-trimethyl-1,3-pentanediol by a recombinant microorganism that comprises an exogenous enzyme having an alcohol dehydrogenase (Adh) activity. Thus, in certain embodiments, a recombinant microorganism comprising one or more polynucleotides encoding an aldolase enzyme and one or more polynucleotides encoding an alcohol dehydrogenase enzyme may be utilized to synthesize 3-hydroxy-2,2,4-trimethyl pentanal and 2,2,4-trimethyl-1,3-pentanediol from a source of isobutyraldehyde, which may then be further converted to isooctane.

For the final steps in the production of isooctane, if desired, 2,2,4-trimethyl-1,3-pentanediol may then be converted to 2,2,4-trimethylpentane, or isooctane, by various processes, such as chemical or enzymatic processes. One example of such a process includes “hydrotreating,” as described herein and known in the art.

As an additional example of converting an aldehyde or ketone to a commodity chemical, a recombinant microorganism provided herein may be utilized to condense two molecules of butyraldehyde to form 3-hydroxy-2-ethylhexanal, which has been shown herein to be catalyzed in vivo by a recombinant microorganism that comprises an exogenous enzyme having an aldolase activity. 3-hydroxy-2-ethylhexanal may then be spontaneously or enzymatically dehydrated to form 2-ethyl-2-hexene-1-al, as also described herein. In certain embodiments, 2-ethyl-2-hexene-1-al may then be consecutively reduced to form 2-ethylhexanal and 2-ethylhexanol, catalyzed by a double bond reductase and an alcohol dehydrogenase, respectively (see Example 4). For the final step, if desired, 2-ethylhexanol may then be converted to 2-ethylhexane according to techniques known in the art and described herein, such as by chemical or enzymatic conversion (e.g., hydrotreating).

Thus, in certain embodiments, a recombinant microorganism comprising one or more exogenous polynucleotides encoding an aldolase enzyme and one or more exogenous polynucleotides encoding an alcohol dehydrogenase enzyme may be utilized to synthesize 2-ethyl-2-hexene-1-al and 2-ethylhexanol from a source of butyraldehyde, which may then be converted to 2-ethylhexane. In certain embodiments, butyraldehyde may be obtained from a recombinant microorganism that comprises a butyraldehyde biosynthesis pathway, as described herein, and which can convert a monosaccharide or oligosaccharide to butyraldehyde.

The methods described herein produce biofuels with significant advantages over other biofuels. In particular, isoctane and other medium to long chain alkanes provide a number of important advantages over the existing common biofuels, such as ethanol and butanol, and are attractive long-term replacements of petroleum-based fuels such as gasoline, diesels, kerosene, and heavy oils in the future. As one example, isooctane and other medium to long chain alkanes and alcohols are major components in all petroleum products and jet fuel in particular, and hence alkanes we produce can be utilized directly by existing engines. By way of further example, medium to long chain alcohols are far better fuels than ethanol, and have a nearly comparable energy density to gasoline. As noted above, isooctane is a major component of gasoline.

As another example, n-alkanes are major components of all oil products including gasoline, diesels, kerosene, and heavy oils. Recombinant microorganisms may be used to produce n-alkanes with different carbon lengths ranging, for example, from C7 to over C20: C7 for gasoline (e.g., motor vehicles), C10-C15 for diesels (e.g., motor vehicles, trains, and ships), and C8-C16 for kerosene (e.g., aviations and ships), and for all heavy oils.

Certain embodiments of the present invention relate generally to methods for producing isooctane and other medium to long chain alkanes from biomass-derived feedstock, thereby providing a low carbon source of biofuels. For instance, in producing a commodity chemical or biofuel (e.g., isooctane) from biomass, a suitable biomass-derived monosaccharide or oligosaccharide may be first obtained directly from any available source, such as a microorganism that is capable of growing on a biomass-derived polysaccharide, such as pectin or alginate, as a sole source of carbon. This monosaccharide or oligosaccharide may then converted to an aldehyde and/or ketone by contacting that monosaccharide or oligosaccharide with a recombinant microorganism that comprises an aldehyde and/or ketone biosynthesis pathway.

The aldehyde and/or ketone produced by such a recombinant microorganism may then be converted to a commodity chemical or biofuel by contacting that aldehyde and/or ketone with a recombinant microorganism of the present invention, such as a microorganism that comprises both an aldolase enzyme and an alcohol dehydrogenase enzyme. In certain embodiments, the recombinant microorganism that comprises the aldehyde and/or ketone biosynthesis pathway may be the same or different as the recombinant microorganism that comprises the aldolase enzyme and the alcohol dehydrogenase enzyme.

Among other uses apparent to a person skilled in the art, the commodity chemicals and biofuels produced by the methods and recombinant microorganisms described herein may be utilized by existing petroleum refineries for blending with petroleum products produced by traditional refinery methods, to produce commodity chemical enriched, refinery-produced petroleum products. To this end, as noted above, fuel producers are seeking substantially similar, low carbon fuels that can be blended and distributed through existing infrastructure (refineries, pipelines, tankers). As hydrocarbons, the commodity chemicals produced according to the methods herein are substantially similar to petroleum derived fuels, reduce green house gas emissions by more than 80% from petroleum derived fuels, and are compatible with existing infrastructure in the oil and gas industry.

For instance, certain of the commodity chemicals produced herein, including, for example, isooctane, among others, are blendable directly into refinery-produced petroleum products, such as gasoline, jet and diesel fuels. By using such biologically produced commodity chemicals as a blendstock for gasoline, jet and diesel fuels, refineries may reduce Green House Gas emissions by more than 80%.

The practice of the present invention will employ, unless indicated specifically to the contrary, conventional methods of molecular biology and recombinant DNA techniques within the skill of the art, many of which are described below for the purpose of illustration. Such techniques are explained fully in the literature. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., 1984); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., 1985); Transcription and Translation (B. Hames & S. Higgins, eds., 1984); A Practical Guide to Molecular Cloning (B. Perbal, ed., 1984).

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below. All references referred to herein are incorporated by reference in their entirety.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

By “about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

The term “biologically active fragment,” as applied to fragments of a reference polynucleotide or polypeptide sequence, refers to a fragment that has at least about 0.1, 0.5, 1, 2, 5, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 100, 110, 120, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000% or more of the activity of a reference sequence.

The term “reference sequence” refers generally to a nucleic acid coding sequence, or amino acid sequence, of any polypeptide or enzyme having a biological activity described herein (e.g., aldolase, alcohol dehydrogenase, dehydratase, diol dehydrogenase, double bond reductase etc.), such as a “wild-type” sequence, including those polynucleotide and polypeptide reference sequences exemplified by SEQ ID NOS:1-96 and 215-222, and including those motif sequences exemplified by SEQ ID NOS:223-260. A reference sequence may also include naturally-occurring, functional variants (i.e., orthologs or homologs) of the sequences described herein.

Included within the scope of the present invention are biologically active fragments of at least about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600 or more contiguous nucleotides or amino acid residues in length, including all integers in between, which comprise or encode a polypeptide having an enzymatic activity of a reference polynucleotide or polypeptide. Representative biologically active fragments generally participate in an interaction, e.g., an intra-molecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction. Examples of enzymatic interactions or activities include aldolase activities, alcohol dehydrogenase activities, dehydratases activities, lyase activities, transporter activities, isomerase activities, kinase activities, among others described herein. Biologically active fragments typically comprise one or more active sites or enzymatic/binding motifs, as described herein and known in the art.

A “biomolecule” refers generally to an organic molecule that is produced by a living organism, including large polymeric molecules (biopolymers) such as proteins, polysaccharides, and nucleic acids as well, as small molecules such as primary secondary metabolites, lipids, phospholipids, glycolipids, sterols, glycerolipids, vitamins, and hormones. Organic molecules (e.g., biomolecules) consist primarily of carbon and hydrogen, nitrogen, and oxygen, and, to a smaller extent, phosphorus and sulfur, although other elements may be incorporated into a biomolecule.

A “biopolymer” refers generally to a large molecule or macromolecule composed of repeating structural units, which are typically connected by covalent chemical bonds, and which can be produced by living organisms. Examples of biopolymers include, without limitation, polysaccharides, nucleic acids, and proteins.

By “coding sequence” is meant any nucleic acid sequence that contributes to the code for the polypeptide product of a gene. By contrast, the term “non-coding sequence” refers to any nucleic acid sequence that does not contribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.

By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules. For example, the sequence “A-G-T,” is complementary to the sequence “T-C-A.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands.

By “corresponds to” or “corresponding to” is meant (a) a polynucleotide having a nucleotide sequence that is substantially identical or complementary to all or a portion of a reference polynucleotide sequence or encoding an amino acid sequence identical to an amino acid sequence in a peptide or protein; or (b) a peptide or polypeptide having an amino acid sequence that is substantially identical to a sequence of amino acids in a reference peptide or protein.

By “derivative” is meant a polypeptide that has been derived from the basic sequence by modification, for example by conjugation or complexing with other chemical moieties (e.g., pegylation) or by post-translational modification techniques as would be understood in the art. The term “derivative” also includes within its scope alterations that have been made to a parent sequence including additions or deletions that provide for functionally equivalent molecules.

By “enzyme reactive conditions” it is meant that any necessary conditions are available in an environment (i.e., such factors as temperature, pH, lack of inhibiting substances) which will permit the enzyme to function. Enzyme reactive conditions can be either in vitro, such as in a test tube, or in vivo, such as within a cell.

As used herein, the terms “function” and “functional” and the like refer to a biological or enzymatic function.

By “gene” is meant a unit of inheritance that occupies a specific locus on a chromosome and consists of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e., introns, 5′ and 3′ untranslated sequences).

“Homology” refers to the percentage number of amino acids that are identical or constitute conservative substitutions. Homology may be determined using sequence comparison programs such as GAP (Deveraux et al., 1984, Nucleic Acids Research 12, 387-395) which is incorporated herein by reference. In this way sequences of a similar or substantially different length to those cited herein could be compared by insertion of gaps into the alignment, such gaps being determined, for example, by the comparison algorithm used by GAP.

The term “host cell” includes an individual cell or cell culture which can be or has been a recipient of any recombinant vector(s) or isolated polynucleotide of the invention. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation and/or change. A host cell includes cells transfected, transformed, or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the invention. A host cell which comprises a recombinant vector of the invention is a recombinant host cell, recombinant cell, or recombinant microrganism.

By “isolated” is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an “isolated polynucleotide”, as used herein, refers to a polynucleotide, which has been purified from the sequences which flank it in a naturally-occurring state, e.g., a DNA fragment which has been removed from the sequences that are normally adjacent to the fragment. Alternatively, an “isolated peptide” or an “isolated polypeptide” and the like, as used herein, refer to in vitro isolation and/or purification of a peptide or polypeptide molecule from its natural cellular environment, and from association with other components of the cell, i.e., it is not associated with in vivo substances.

By “enhance,” “enhancing,” “increase,” or “increasing” is meant the ability of one or more recombinant microorganisms to produce a greater amount of a given product or molecule (e.g., commodity chemical, biofuel, or intermediate product thereof) as compared to a control microorganism, such as an unmodified microorganism or a differently modified microorganism. An “increased” amount is typically a “statistically significant” amount, and may include an increase that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more times (including all integers and decimal points in between, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by an unmodified microorganism or a differently modified microorganism. An increased amount may be measured according to routine techniques in the art. For instance, an “increased” amount of a commodity chemical may be measured according to a percentage of a theoretical maximum yield. For instance, in certain embodiments, the methods of the present invention may enhance the yield of a target molecule (e.g., commodity chemical) to at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of a theoretical maximum yield. In certain embodiments, the method may be characterized by increasing the percentage of the theoretical maximum yield of the target molecule by at least about 10% (e.g., from about 30% to about 40% of the theoretical maximum yield), 15% (e.g., from about 30% to about 45%), 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, or 90% as compared to incubating the same recombinant microorganism under control or different conditions, or as compared to incubating a control (e.g., unmodified or differently modified) microorganism under the same or similar conditions.

The term “reduce” relates generally to a “decrease” in a relevant cellular response, such as NADH or acetate production, as measured according to routine techniques in the diagnostic art. Other relevant cellular responses (in vivo or in vitro) will be apparent to persons skilled in the art. A “decrease” in a response may be “statistically significant” amount as compared to the response produced by an unmodified microorganism or a differently modified microorganism, or by a microorganism growing under different conditions, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in between.

By “obtained from” is meant that a sample such as, for example, a polynucleotide extract or polypeptide extract is isolated from, or derived from, a particular source, such as a desired organism, typically a microorganism. “Obtained from” can also refer to the situation in which a polynucleotide or polypeptide sequence is isolated from, or derived from, a particular organism or microorganism. For example, a polynucleotide sequence encoding a benzaldehyde lyase enzyme may be isolated from a variety of prokaryotic or eukaryotic microorganisms, such as Pseudomonas. As another example, a polynucleotide sequence encoding an aldolase enzyme may be isolated from a variety of prokaryotic or eukarotic microrganisms, such as Thermotoga maritima and Escherichia coli DH10B.

The term “operably linked” as used herein means placing a gene under the regulatory control of a promoter, which then controls the transcription and optionally the translation of the gene. In the construction of heterologous promoter/structural gene combinations, it is generally preferred to position the genetic sequence or promoter at a distance from the gene transcription start site that is approximately the same as the distance between that genetic sequence or promoter and the gene it controls in its natural setting; i.e. the gene from which the genetic sequence or promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting; i.e., the genes from which it is derived. “Constitutive promoters” are typically active, i.e., promote transcription, under most conditions. “Inducible promoters” are typically active only under certain conditions, such as in the presence of a given molecule factor (e.g., IPTG) or a given environmental condition (e.g., CO₂ concentration, nutrient levels, light, heat). In the absence of that condition, inducible promoters typically do not allow significant or measurable levels of transcriptional activity.

The recitation “polynucleotide” or “nucleic acid” as used herein designates mRNA, RNA, cRNA, rRNA, cDNA or DNA. The term typically refers to polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms of DNA.

As will be understood by those skilled in the art, the polynucleotide sequences of this invention can include genomic sequences, extra-genomic and plasmid-encoded sequences and smaller engineered gene segments that express, or may be adapted to express, proteins, polypeptides, peptides and the like. Such segments may be naturally isolated, or modified synthetically by the hand of man.

Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA or synthetic) or RNA molecules. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

Polynucleotides may comprise a native sequence or may comprise a variant, or a biological functional equivalent of such a sequence. Polynucleotide variants may contain one or more substitutions, additions, deletions and/or insertions, as further described below, preferably such that the enzymatic activity of the encoded polypeptide is not substantially diminished relative to the unmodified polypeptide, and preferably such that the enzymatic activity of the encoded polypeptide is improved (e.g., optimized) relative to the unmodified polypeptide. The effect on the enzymatic activity of the encoded polypeptide may generally be assessed as described herein.

In certain embodiments, the present invention provides isolated polynucleotides comprising various lengths of contiguous stretches of sequence identical to or complementary to an aldolase or an alcohol dehydrogenase, among others described herein, wherein the isolated polynucleotides encode a biologically active, truncated enzyme.

Exemplary nucleotide sequences that encode the enzymes of the application encompass full-length aldolases and alcohol dehydrogenases, as well as portions of the full-length or substantially full-length nucleotide sequences of these genes or their transcripts or DNA copies of these transcripts. Portions of a nucleotide sequence may encode polypeptide portions or segments that retain the biological activity of the reference polypeptide. A portion of a nucleotide sequence that encodes a biologically active fragment of an enzyme provided herein may encode at least about 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500, 600, or more contiguous amino acid residues, almost up to the total number of amino acids present in a full-length enzyme. It will be readily understood that “intermediate lengths,” in this context and in all other contexts used herein, means any length between the quoted values, such as 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

The polynucleotides of the present invention, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a polynucleotide fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant DNA protocol.

The terms “polynucleotide variant” and “variant” and the like refer to polynucleotides that display substantial sequence identity with any of the reference polynucleotide sequences or genes described herein, and to polynucleotides that hybridize with any polynucleotide reference sequence described herein, or any polynucleotide coding sequence of any gene or polypeptide referred to herein, under low stringency, medium stringency, high stringency, or very high stringency conditions that are defined hereinafter and known in the art. These terms also encompass polynucleotides that are distinguished from a reference polynucleotide by the addition, deletion or substitution of at least one nucleotide. Accordingly, the terms “polynucleotide variant” and “variant” include polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference polynucleotide whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide, or has increased activity in relation to the reference polynucleotide (i.e., optimized). Polynucleotide variants include, for example, polynucleotides having at least 50% (and at least 51% to at least 99% and all integer percentages in between) sequence identity with a reference polynucleotide described herein.

The terms “polynucleotide variant” and “variant” also include naturally-occurring allelic variants that encode these enzymes. Examples of naturally-occurring variants include allelic variants (same locus), homologs (different locus), and orthologs (different organism). Naturally occurring variants such as these can be identified and isolated using well-known molecular biology techniques including, for example, various polymerase chain reaction (PCR) and hybridization-based techniques as known in the art. Naturally occurring variants can be isolated from any organism that encodes one or more genes having a suitable enzymatic activity described herein (e.g., C—C ligase, aldolase, alcohol dehydrogenase, reductase, etc.).

Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. In certain aspects, non-naturally occurring variants may have been optimized for use in a given microorganism (e.g., E. coli), such as by engineering and screening the enzymes for increased activity, stability, or any other desirable feature. The variations can produce both conservative and non-conservative amino acid substitutions (as compared to the originally encoded product). For nucleotide sequences, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of a reference polypeptide. Variant nucleotide sequences also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a biologically active polypeptide.

Generally, variants of a particular reference nucleotide sequence may have at least about 30%, 40% 50%, 55%, 60%, 65%, 70%, generally at least about 75%, 80%, 85%, 90% to 95% or more, and even about 97% or 98% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein using default parameters.

Known aldolase, alcohol dehydrogenase, double bond reductase, and other nucleotide reference sequences can be used to isolate corresponding sequences and alleles from other organisms, particularly other microorganisms. Methods are readily available in the art for the hybridization of nucleic acid sequences. Coding sequences from other organisms may be isolated according to well known techniques based on their sequence identity with the coding sequences set forth herein. In these techniques all or part of the known coding sequence is used as a probe which selectively hybridizes to other reference coding sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism.

The present invention also contemplates polynucleotides that hybridize to reference aldolase, alcohol dehydrogenase, double bond reductase, or other nucleotide sequences, or to their complements, under stringency conditions described below. As used herein, the term “hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley & Sons Inc, 1994-1998, Sections 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used.

Reference herein to “low stringency” conditions include and encompass from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization at 42° C., and at least about 1 M to at least about 2 M salt for washing at 42° C. Low stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at room temperature. One embodiment of low stringency conditions includes hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes can be increased to 55° C. for low stringency conditions).

“Medium stringency” conditions include and encompass from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization at 42° C., and at least about 0.1 M to at least about 0.2 M salt for washing at 55° C. Medium stringency conditions also may include 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS for washing at 60-65° C. One embodiment of medium stringency conditions includes hybridizing in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.

“High stringency” conditions include and encompass from at least about 31% v/v to at least about 50% v/v formamide and from about 0.01 M to about 0.15 M salt for hybridization at 42° C., and about 0.01 M to about 0.02 M salt for washing at 55° C. High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a temperature in excess of 65° C. One embodiment of high stringency conditions includes hybridizing in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.

One embodiment of “very high stringency” conditions includes hybridizing in 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes in 0.2×SSC, 1% SDS at 65° C.

Other stringency conditions are well known in the art and a skilled addressee will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization. For detailed examples, see Ausubel et al., supra at pages 2.10.1 to 2.10.16 and Sambrook et al., Current Protocols in Molecular Biology (1989), at sections 1.101 to 1.104.

While stringent washes are typically carried out at temperatures from about 42° C. to 68° C., one skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization rate typically occurs at about 20° C. to 25° C. below the T_(m) for formation of a DNA-DNA hybrid. It is well known in the art that the T_(m) is the melting temperature, or temperature at which two complementary polynucleotide sequences dissociate. Methods for estimating T_(m) are well known in the art (see Ausubel et al., supra at page 2.10.8).

In general, the T_(m) of a perfectly matched duplex of DNA may be predicted as an approximation by the formula: T_(m)=81.5+16.6 (log₁₀ M)+0.41 (% G+C)−0.63 (% formamide)−(600/length) wherein: M is the concentration of Na⁺, preferably in the range of 0.01 molar to 0.4 molar; % G+C is the sum of guano sine and cytosine bases as a percentage of the total number of bases, within the range between 30% and 75% G+C; % formamide is the percent formamide concentration by volume; length is the number of base pairs in the DNA duplex. The T_(m) of a duplex DNA decreases by approximately 1° C. with every increase of 1% in the number of randomly mismatched base pairs. Washing is generally carried out at T_(m)−15° C. for high stringency, or T_(m)−30° C. for moderate stringency.

In one example of a hybridization procedure, a membrane (e.g., a nitrocellulose membrane or a nylon membrane) containing immobilized DNA is hybridized overnight at 42° C. in a hybridization buffer (50% deionizer formamide, 5×SSC, 5× Reinhardt's solution (0.1% fecal, 0.1% polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200 mg/mL denatured salmon sperm DNA) containing a labeled probe. The membrane is then subjected to two sequential medium stringency washes (i.e., 2×SSC, 0.1% SDS for 15 min at 45° C., followed by 2×SSC, 0.1% SDS for 15 min at 50° C.), followed by two sequential higher stringency washes (i.e., 0.2×SSC, 0.1% SDS for 12 min at 55° C. followed by 0.2×SSC and 0.1% SDS solution for 12 min at 65-68° C. Based on the above, embodiments of the present invention include recombinant microorganisms that comprise a polynucleotide sequence that hybridizes to the complement of any of the reference polynucleotide sequences described herein, such as a polynucleotide sequence that encodes an aldolase or an alcohol dehydrogenase enzyme, under conditions of medium, high, or very high stringency, as described herein and known in the art.

Polynucleotides and fusions thereof may be prepared, manipulated and/or expressed using any of a variety of well established techniques known and available in the art. For example, polynucleotide sequences which encode polypeptides of the invention, or fusion proteins or functional equivalents thereof, may be used in recombinant DNA molecules to direct expression of a selected enzyme in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences that encode substantially the same or a functionally equivalent amino acid sequence may be produced and these sequences may be used to clone and express a given polypeptide.

As will be understood by those of skill in the art, it may be advantageous in some instances to produce polypeptide-encoding nucleotide sequences possessing non-naturally occurring codons. For example, codons preferred by a particular prokaryotic or eukaryotic host can be selected to increase the rate of protein expression or to produce a recombinant RNA transcript having desirable properties, such as a half-life that is longer than that of a transcript generated from the naturally occurring sequence. Such nucleotides are typically referred to as “codon-optimized.” Any of the nucleotide sequences described herein may be utilized in such a “codon-optimized” form.

Moreover, the polynucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter polypeptide encoding sequences for a variety of reasons, including but not limited to, alterations which modify the cloning, processing, expression and/or activity of the gene product.

In order to express a desired polypeptide, a nucleotide sequence encoding the polypeptide, or a functional equivalent, may be inserted into appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods which are well known to those skilled in the art may be used to construct expression vectors containing sequences encoding a polypeptide of interest and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989).

Host cells transformed with a polynucleotide sequence of interest may be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides of the invention may be designed to contain signal sequences which direct localization of the encoded polypeptide to a desired site within the cell. Other recombinant constructions may be used to join sequences encoding a polypeptide of interest to nucleotide sequence encoding a polypeptide domain which will direct secretion of the encoded protein.

Embodiments of the present invention contemplate the use of recombinant microorganisms comprising “polypeptides” having an aldolase activity, alcohol dehydrogenase activity, double bond reductase activity, or other activity described herein, including truncated, variant and/or modified polypeptides thereof, for producing commodity chemicals. “Polypeptide,” “polypeptide fragment,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers. In certain aspects, polypeptides may include enzymatic polypeptides, or “enzymes,” which typically catalyze (i.e., increase the rate of) various chemical reactions.

The recitation polypeptide “variant” refers to polypeptides that are distinguished from a reference polypeptide sequence by the addition, deletion or substitution of at least one amino acid residue. In certain embodiments, a polypeptide variant may be distinguished from a reference polypeptide by one or more substitutions, which may be conservative or non-conservative. In certain embodiments, the polypeptide variant comprises conservative substitutions and, in this regard, it is well understood in the art that some amino acids may be changed to others with broadly similar properties without changing the nature of the activity of the polypeptide. Polypeptide variants also encompass polypeptides in which one or more amino acids have been added or deleted, or replaced with different amino acid residues.

Variant proteins encompassed by the present application are “biologically active,” that is, they continue to possess the enzymatic activity of a reference polypeptide. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a reference polypeptide sequence or fragment thereof may have at least about 40%, 50%, 60%, 70%, generally at least about 75%, 80%, 85%, usually about 90% to 95% or more, and typically about 98% or more sequence similarity or identity with the amino acid sequence for a reference protein as determined by sequence alignment programs described elsewhere herein using default parameters. A biologically active variant of a reference polypeptide may differ from that protein generally by as much 200, 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. In some embodiments, a variant polypeptide differs from a reference polypeptide sequences by at least one but by less than 15, 10 or 5 amino acid residues. In other embodiments, it differs from the reference sequences by at least one residue but less than 20%, 15%, 10% or 5% of the residues.

The present invention contemplates the use in the methods described herein of variants of full-length reference polypeptides having any of the enzymatic activities described herein (e.g., aldolase, alcohol dehydrogenase, double bond reductase etc.), truncated fragments of these full-length polypeptides, variants of truncated fragments, as well as their related biologically active fragments. Typically, biologically active fragments of a polypeptide may participate in an interaction, for example, an intra-molecular or an inter-molecular interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). Biologically active fragments of a polypeptide/enzyme an enzymatic activity described herein include peptides comprising amino acid sequences sufficiently similar to, or derived from, the amino acid sequences of a (putative) full-length reference polypeptide sequence. Typically, biologically active fragments comprise a domain or motif with at least one enzymatic activity, and may include one or more (and in some cases all) of the various active domains. A biologically active fragment of an enzyme can be a polypeptide fragment which is, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 450, 500, 600 or more contiguous amino acids, including all integers in between, of a reference polypeptide sequence. In certain embodiments, a biologically active fragment comprises a conserved enzymatic sequence, domain, or motif, as described elsewhere herein and known in the art. Suitably, the biologically-active fragment has no less than about 1%, 10%, 25%, 50% of an activity of the wild-type polypeptide from which it is derived.

An aldolase, alcohol dehydrogenase, double bond reductase, or other reference polypeptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S. Pat. No. 4,873,192, Watson, J. D. et al., (“Molecular Biology of the Gene”, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of aldolase or alcohol dehydrogenase polypeptides. Recursive ensemble mutagenesis (REM), a technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify polypeptide variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave et al., (1993) Protein Engineering, 6: 327-331). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below.

Polypeptide variants may contain conservative amino acid substitutions at various locations along their sequence, as compared to a reference amino acid sequence. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub-classified as follows:

Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.

This description also characterizes certain amino acids as “small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, “small” amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the α-amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAM120 matrix and PAM250 matrix as disclosed for example by Dayhoff et al., (1978), A model of evolutionary change in proteins. Matrices for determining distance relationships In M. O. Dayhoff, (ed.), Atlas of protein sequence and structure, Vol. 5, pp. 345-358, National Biomedical Research Foundation, Washington D.C.; and by Gonnet et al., (Science, 256: 14430-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a “small” amino acid.

The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary and, therefore, amino acids specifically contemplated by the invention have been classified as one or the other. Most amino acids not specifically named can be classified on the basis of known behaviour.

Amino acid residues can be further sub-classified as cyclic or non-cyclic, and aromatic or non-aromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always non-aromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in Table A.

TABLE 1 Amino acid sub-classification Sub-classes Amhio acids Acidic Aspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic: Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine, Histidine Small Glycine, Serine, Alanine, Threonine, Proline Polar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine, Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine, Valine, Isoleucine, Leucine, Methionine, Phenylalanine, Tryptophan Aromatic Tryptophan, Tyrosine, Phenylalanine Residues that influence Glycine and Proline chain orientation

Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional truncated and/or variant polypeptide can readily be determined by assaying its enzymatic activity, as described herein (see, e.g., Examples 2-4). Conservative substitutions are shown in Table B under the heading of exemplary substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

TABLE 2 Exemplary Amino Acid Substitutions Original Residue Exemplary Substitutions Preferred Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn, His, Lys, Asn Glu Asp, Lys Asp Gly Pro Pro His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleu Leu Leu Norleu, Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleu Leu

Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm. C. Brown Publishers (1993).

Thus, a predicted non-essential amino acid residue in an aldolase or alcohol dehydrogenase polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially abolish one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% 100%, 500%, 1000% or more of wild-type. An “essential” amino acid residue is a residue that, when altered from the wild-type sequence of a reference polypeptide, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present. For example, such essential amino acid residues include those that are conserved in aldolase, alcohol dehydrogenase, double bond reductase, or other reference polypeptides across different species, including those sequences that are conserved in the enzymatic sites of polypeptides from various sources.

Accordingly, the present invention also contemplates variants of the naturally-occurring reference polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% similarity or sequence identity to a reference polypeptide sequence. Moreover, sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60,70, 80, 90, 100 or more amino acids but which retain the properties of a parent or reference polypeptide sequence are contemplated.

In some embodiments, variant polypeptides differ from a reference aldolase or alcohol dehydrogenase polypeptide sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In other embodiments, variant polypeptides differ from a reference by at least 1% but less than 20%, 15%, 10% or 5% of the residues. (If this comparison requires alignment, the sequences should be aligned for maximum similarity. “Looped” out sequences from deletions or insertions, or mismatches, are considered differences.)

In certain embodiments, a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, or 99% or more sequence identity or similarity to a corresponding sequence of a reference polypeptide, and retains the enzymatic activity of that reference polypeptide.

The recitations “sequence identity” or, for example, comprising a “sequence 50% identical to,” as used herein, refer to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, H is, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence identity”, “percentage of sequence identity” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of at least 6 contiguous positions, usually about 50 to about 100, more usually about 100 to about 150 in which a sequence is compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The comparison window may comprise additions or deletions (i.e., gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.

Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e., resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al., “Current Protocols in Molecular Biology”, John Wiley & Sons Inc, 1994-1998, Chapter 15.

The term “endogenous” refers generally to naturally occurring copies of a gene, polynucleotide sequence, or polypeptide that may be found in a genetically unmodified wild-type cell or organism. For example, certain naturally-occurring bacterial or yeast species do not typically contain an aldolase gene, and, therefore, do not comprise an “endogenous” polynucleotide sequence that encodes a aldolase enzyme.

The term “exogenous” refers generally to a copy of a gene, copy of polynucleotide sequence or nucleic acid molecule, or a polypeptide that does not naturally occur in a wild-type cell or organism, but is typically introduced into the cell by molecular biological techniques, i.e., engineering to produce a recombinant microorganism. Examples of “exogenous” genes or polynucleotides include vectors, plasmids, and/or man-made nucleic acid constructs encoding a desired protein or enzyme.

In this regard, it is also noted that even though an organism may comprise an endogenous or naturally-occurring copy of a given polynucleotide sequence or gene, the introduction of a plasmid or vector encoding that sequence or a related sequence, such as to over-express or otherwise regulate the expression of the encoded polypeptide, represents an “exogenous” copy of that gene or polynucleotide sequence. Also, in certain embodiments, the placing of an otherwise endogenous gene or polynucleotide under the control of an exogenous promoter sequence, such as a constitutive or inducible promoter, to increase the level of expression of the endogenous gene, may also be considered an exogenous gene or polynucleotide within the meaning of the present invention. Similarly, the modification of an otherwise endogenous gene or polynucleotide by the addition of non-naturally occurring sequences, such as to produce a chimeric gene or polypeptide, may also be considered an exogenous gene or polynucleotide within the meaning of the present invention.

Any of the of pathways, genes, polynucleotides, nucleic acid molecules, polypeptides, or enzymes described herein may utilize or rely on an “endogenous” sequence, or may be provided as one or more “exogenous” sequences.

The present invention also contemplates chimeric or fusion polypeptides. As used herein, a “chimeric protein,” “fusion protein,” or “fusion polypeptide” may include, without limitation, a first polypeptide or fragment thereof linked to a second, third, or fourth (or more) polypeptide, or fragment thereof (e.g., to create multiple fragments). The second, third or fourth polypeptide may refer to the same polypeptide as the first polypeptide, such as to selectively link together certain fragments of that first polypeptide, or may refer to a “heterologous polypeptide,” which typically has an amino acid sequence corresponding to a protein that is different from the first polypeptide, and which may be derived from the same or a different organism. In certain embodiments, a fusion protein includes at least one (or two, three, four, or more) biologically active portion of a given polypeptide protein. The polypeptides forming the fusion protein are typically linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of the fusion protein can be in any order.

The fusion partner may be designed and included for essentially any desired purpose provided they do not adversely affect the activity of the polypeptide. For example, in one embodiment, a fusion partner may comprise a sequence that assists in expressing the protein (an expression enhancer) at higher yields than the native recombinant protein. Other fusion partners may be selected so as to increase the solubility of the protein, target the protein to desired intracellular compartments, secrete the protein, or tether the protein to the cell surface. As one example, the fusion protein can contain a heterologous signal peptide sequence at its N-terminus. In certain host cells, secretion or cell-surface tethering of fusion polypeptides can be increased through the use of one or more heterologous signal peptide sequences, typically fused at or near to the N-terminus of the polypeptide.

A “recombinant” microorganism typically comprises one or more exogenous genes or polynucleotide sequences, such as in a plasmid or vector. Examples of microorganisms that can be utilized as recombinant microorganisms include, without limitation, Escherichia coli, Acetobacter aceti, Achromobacter, Acidiphilium, Acinetobacter, Actinomadura, Actinoplanes, Aeropyrum pernix, Agrobacterium, Alcaligenes, Ananas comosus (M), Arthrobacter, Aspargillus niger, Aspargillus oryze, Aspergillus melleus, Aspergillus pulverulentus, Aspergillus saitoi, Aspergillus sojea, Aspergillus usamii, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus lentus, Bacillus licheniformis, Bacillus macerans, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium, Brevibacillus brevis, Burkholderia cepacia, Candida cylindracea, Candida rugosa, Carica papaya (L), Cellulosimicrobium, Cephalosporium, Chaetomium erraticum, Chaetomium gracile, Clostridium, Clostridium butyricum, Clostridium acetobutylicum, Clostridium thermocellum, Corynebacterium (glutamicum), Corynebacterium efficiens, Enterococcus, Erwina chrysanthemi, Gliconobacter, Gluconacetobacter, Haloarcula, Humicola insolens, Humicola nsolens, Kitasatospora setae, Klebsiella, Klebsiella oxytoca, Kluyveromyces, Kluyveromyces fragilis, Kluyveromyces lactis, Kocuria, Lactlactis, Lactobacillus, Lactobacillus fermentum, Lactobacillus sake, Lactococcus, Lactococcus lactis, Leuconostoc, Methylocystis, Methanolobus siciliae, Methanogenium organophilum, Methanobacterium bryantii, Microbacterium imperiale, Micrococcus lysodeikticus, Microlunatus, Mucor javanicus, Mycobacterium, Myrothecium, Nitrobacter, Nitrosomonas, Nocardia, Papaya carica, Pediococcus, Pediococcus halophilus, Penicillium, Penicillium camemberti, Penicillium citrinum, Penicillium emersonii, Penicillium roqueforti, Penicillum lilactinum, Penicillum multicolor, Paracoccus pantotrophus, Propionibacterium, Pseudomonas, Pseudomonas fluorescens, Pseudomonas denitrificans, Pyrococcus, Pyrococcus furiosus, Pyrococcus horikoshii, Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Rhizopus oryzae, Rhizopus oligosporus, Rhodococcus, Saccharophagus degradans, Sccharomyces cerevisiae, Sclerotina libertina, Sphingobacterium multivorum, Sphingobium, Sphingomonas, Streptococcus, Streptococcus thermophilus Y-1, Streptomyces, Streptomyces griseus, Streptomyces lividans, Streptomyces murinus, Streptomyces rubiginosus, Streptomyces violaceoruber, Streptoverticillium mobaraense, Tetragenococcus, Thermus, Thiosphaera pantotropha, Trametes, Trichoderma, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon penicillatum, Vibrio alginolyticus, Vibrio splendidus, Xanthomonas, yeast, Yarrowia lipolytica, Zygosaccharomyces rouxii, Zymomonas, or Zymomonus mobilis.

“Transformation” refers generally to the permanent, heritable alteration in a cell resulting from the uptake and incorporation of foreign DNA into the host-cell genome; also, the transfer of an exogenous gene from one organism into the genome of another organism.

By “vector” is meant a polynucleotide molecule, preferably a DNA molecule derived, for example, from a plasmid, bacteriophage, yeast or virus, into which a polynucleotide can be inserted or cloned. A vector preferably contains one or more unique restriction sites and can be capable of autonomous replication in a defined host cell including a target cell or tissue or a progenitor cell or tissue thereof, or be integrable with the genome of the defined host such that the cloned sequence is reproducible. Accordingly, the vector can be an autonomously replicating vector, i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, e.g., a linear or closed circular plasmid, an extra-chromosomal element, a mini-chromosome, or an artificial chromosome. The vector can contain any means for assuring self-replication. Alternatively, the vector can be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Such a vector may comprise specific sequences that allow recombination into a particular, desired site of the host chromosome.

A vector system can comprise a single vector or plasmid, two or more vectors or plasmids, which together contain the total DNA to be introduced into the genome of the host cell, or a transposon. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. In the present case, the vector is preferably one which is operably functional in a bacterial cell, such as a cyanobacterial cell. The vector can include a reporter gene, such as a green fluorescent protein (GFP), which can be either fused in frame to one or more of the encoded polypeptides, or expressed separately. The vector can also include a selection marker such as an antibiotic resistance gene that can be used for selection of suitable transformants.

The terms “wild-type” and “naturally occurring” are used interchangeably to refer to a gene or gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild type gene or gene product (e.g., a polypeptide) is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene.

Examples of “biomass” include aquatic or marine biomass, fruit-based biomass such as fruit waste, and vegetable-based biomass such as vegetable waste, among others. Examples of aquatic or marine biomass include, but are not limited to, kelp, giant kelp, seaweed, algae, and marine microflora, microalgae, sea grass, and the like. Examples of fruit and/or vegetable biomass include, but are not limited to, any source of pectin such as plant peel and pomace including citrus, orange, grapefruit, potato, tomato, grape, mango, gooseberry, carrot, sugar-beet, and apple, among others. In certain aspects, biomass does not include fossilized sources of carbon, such as hydrocarbons that are typically found within the top layer of the Earth's crust (e.g., natural gas, nonvolatile materials composed of almost pure carbon, like anthracite coal, etc).

A “commodity chemical,” or an “intermediate thereof,” relates generally to chemicals, such as biofuels, having the following formulas:

wherein R1 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, and CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂; and

wherein R2 is selected from the group consisting of H, CH₃, CH₃CH₂, CH₃CH(CH₃), CH₃(CH₂)_(n)CH₂, CH₃CH(CH₃)(CH₂)_(n)CH₂, CH₃CH₂CH(CH₃)(CH₂)_(n)CH₂,

wherein n=0-30, including any corresponding alkanes that can be produced therefrom.

In certain embodiments, the commodity chemical may be 3-hydroxy-2,2,4 trimethylpentanal, 2,2,4-trimethyl-1,3-pentanediol, and/or 2,2,4-trimethylpentane. In certain embodiments, the commodity chemical may be 3-hydroxy-2-ethylhexanal, 2-ethyl-2-hexene-1-al, 2-ethylhexanal, 2-ethylhexanol, and/or 2-ethylhexane. In certain embodiments, the commodity chemical may be 3-hydroxy-2-butyl-1-octanol, 2-butyl-2-octene-1-al, 2-butyl-octanal, or 2-butyl-octanol.

In certain aspects, the diol or alcohol, such as 2,2,4-trimethyl-1,3-pentanediol or 2-ethylhexanol, may be further chemically or enzymatically converted to its corresponding alkane, such as 2,2,4-trimethylpentane or 2-ethylhexane, respectively. One example of such a process includes “hydrotreating,” also known as “hydrogenation.” For instance, alcohols such as 2,2,4-trimethyl-1,3-pentanediol, 2-ethylhexanol, and 2-butyl-octanol may be “hydrotreated” to form their corresponding alkanes, 2,2,4-trimethylpentane, 2-ethylhexane, and 2-butyl-octane, respectively. The process of “hydrotreating” in petroleum refining systems refers generally to the catalytic addition of hydrogen to a molecule containing a functional group. A hydrotreating catalyst is generally formulated by placing some combination of molybdenum, nickel and cobalt oxide on a calcined alumina particle. The alumina particle may then be formulated and manufactured to have an extremely high internal surface area (very porous) and the catalytic metals may be deposited and distributed “mono-atomically” on the internal surface of the particle. In hydrotreating (vs. “hydrocracking”) the alumina surface is typically formulated and manufactured to be as inert (i.e., chemically passive) as possible.

General examples of “biomass-derived polysaccharides” include, but are not limited to, alginate, agar, carrageenan, fucoidan, pectin, polygalacturonate, cellulose, hemicellulose, xylan, arabinan, and mannan. Examples of polysaccharides, oligosaccharides, monosaccharides or other sugar components of biomass include, but are not limited to, alginate (e.g., polyG, polyMG, polyM), oligoalginate (e.g., ΔM, ΔG, ΔMM, ΔMG, ΔGM, ΔGG, MM, MG, GM, GG, MMM, MGM, MMG, MGG, GMM, GMG, GGM, GGG), agar, carrageenan, fucoidan, pectin, gluronate, guluronate, mannuronate, mannitol, lyxose, cellulose, hemicellulose, cellobiose, glycerol, xylitol, glucose, mannose, galactose, xylose, xylan, mannan, arabinan, arabinose, glucuronate, galacturonate (including di- and tri-galacturonates), rhamnose, and the like.

Certain examples of alginate-derived polysaccharides include saturated polysaccharides, such as β-D-mannuronate, α-L-gluronate, dialginate, trialginate, pentalginate, hexylginate, heptalginate, octalginate, nonalginate, decalginate, undecalginate, dodecalginate and polyalginate, as well as unsaturated polysaccharides such as 4-deoxy-L-erythro-5-hexoseulose uronic acid, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-D-mannuronate or L-guluronate, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-dialginate, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-trialginate, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-tetralginate, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-pentalginate, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-hexylginate, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-heptalginate, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-octalginate, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-non alginate, 4-(4-deoxy-beta-D-mann-4-enuronosyl)-undecalginate, and 4-(4-deoxy-beta-D-mann-4-enuronosyl)-dodecalginate.

Certain examples of pectin-derived polysaccharides include saturated polysaccharides, such as galacturonate, digalacturonate, trigalacturonate, tetragalacturonate, pentagalacturonate, hexagalacturonate, heptagalacturonate, octagalacturonate, nonagalacturonate, decagalacturonate, dodecagalacturonate, polygalacturonate, and rhamnopolygalacturonate, as well as saturated polysaccharides such as 4-deoxy-L-threo-5-hexosulose uronate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-galacturonate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-digalacturonate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-trigalacturonate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-tetragalacturonate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-pentagalacturonate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-hexagalacturonate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-heptagalacturonate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-octagalacturonate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-nonagalacturonate, 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-decagalacturonate, and 4-(4-Deoxy-alpha-D-gluc-4-enuronosyl)-D-dodecagalacturonate.

A “suitable monosaccharide” or “suitable saccharide” refers generally to any saccharide that may be produced by a recombinant microorganism growing on pectin, alginate, or other saccharide (e.g., biomass-derived polysaccharides including galacturonate, cellulose, hemi-cellulose etc.) as a source or sole source of carbon, and also refers generally to any saccharide that may be utilized in a biofuel biosynthesis pathway of the present invention to produce hydrocarbons such as biofuels or biopetrols. Examples of suitable monosaccharides or oligosaccharides include, but are not limited to, 2-keto-3-deoxy D-gluconate (KDG), D-mannitol, gluronate, mannuronate, mannitol, lyxose, glycerol, xylitol, glucose, mannose, galactose, xylose, arabinose, glucuronate, galacturonates, and rhamnose, and the like. As noted herein, a “suitable monosaccharide” or “suitable saccharide” as used herein may be produced by an engineered or recombinant microorganism described in U.S. application Ser. Nos. 12/245,537 and 12/245,540, herein incorporated by reference for the disclosure of such microorganisms, or may be obtained from commercially available sources.

The recitation “optimized” as used herein refers to a pathway, gene, polypeptide, enzyme, or other molecule having an altered biological activity, such as by the genetic alteration of a polypeptide's amino acid sequence or by the alteration/modification of the polypeptide's surrounding cellular environment, to improve its functional characteristics in relation to the original molecule or original cellular environment (e.g., a wild-type sequence of a given polypeptide or a wild-type microorganism). Any of the polypeptides or enzymes described herein may be optionally “optimized,” and any of the genes or nucleotide sequences described herein may optionally encode an optimized polypeptide or enzyme. Any of the pathways described herein may optionally contain one or more “optimized” enzymes, or one or more nucleotide sequences encoding for an optimized enzyme or polypeptide.

Typically, the improved functional characteristics of the polypeptide, enzyme, or other molecule relate to the suitability of the polypeptide or other molecule for use in a biological pathway (e.g., an aldehyde and/or ketone biosynthesis pathway, an aldolase, an alcohol dehydrogenase) to convert an aldehyde and/or ketone to a biofuel, such as isooctane. Certain embodiments, therefore, contemplate the use of “optimized” biological pathways. An exemplary “optimized” polypeptide may contain one or more alterations or mutations in its amino acid coding sequence (e.g., point mutations, deletions, addition of heterologous sequences) that facilitate improved expression and/or stability in a given microbial system or microorganism, allow regulation of polypeptide activity in relation to a desired substrate (e.g., inducible or repressible activity), modulate the localization of the polypeptide within a cell (e.g., intracellular localization, extracellular secretion), and/or effect the polypeptide's overall level of activity in relation to a desired substrate (e.g., reduce or increase enzymatic activity). A polypeptide or other molecule may also be “optimized” for use with a given microbial system or microorganism by altering one or more pathways within that system or organism, such as by altering a pathway that regulates the expression (e.g., up-regulation), localization, and/or activity of the “optimized” polypeptide or other molecule, or by altering a pathway that minimizes the production of undesirable by-products, among other alterations. In this manner, a polypeptide or other molecule may be “optimized” with or without altering its wild-type amino acid sequence or original chemical structure. Optimized polypeptides or biological pathways may be obtained, for example, by direct mutagenesis or by natural selection for a desired phenotype, according to techniques known in the art.

In certain aspects, “optimized” genes or polypeptides may comprise a nucleotide coding sequence or amino acid sequence that is 50% to 99% identical (including all integers in between) to the nucleotide or amino acid sequence of a reference (e.g., wild-type) gene or polypeptide. In certain aspects, an “optimized” polypeptide or enzyme may have about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100 (including all integers and decimal points in between e.g., 1.2, 1.3, 1.4, 1.5, 5.5, 5.6, 5.7, 60, 70, etc.), or more times the biological activity of a reference polypeptide.

Certain aspects of the invention also include a commodity chemical, such as a biofuel, that is produced according to the methods and recombinant microorganisms described herein. Such a biofuel (e.g., medium to long chain alkane, isooctane) may be distinguished from other fuels, such as those fuels produced by traditional refinery from crude carbon sources, by radio-carbon dating techniques. For instance, carbon has two stable, nonradioactive isotopes: carbon-12 (¹²C), and carbon-13 (¹³C). In addition, there are trace amounts of the unstable isotope carbon-14 (¹⁴C) on Earth. Carbon-14 has a half-life of 5730 years, and would have long ago vanished from Earth were it not for the unremitting impact of cosmic rays on nitrogen in the Earth's atmosphere, which create more of this isotope. The neutrons resulting from the cosmic ray interactions participate in the following nuclear reaction on the atoms of nitrogen molecules (N₂) in the atmospheric air:

n+ ₇ ¹⁴N→₆ ¹⁴C+p

Plants and other photosynthetic organisms take up atmospheric carbon dioxide by photosynthesis. Since many plants are ingested by animals, every living organism on Earth is constantly exchanging carbon-14 with its environment for the duration of its existence. Once an organism dies, however, this exchange stops, and the amount of carbon-14 gradually decreases over time through radioactive beta decay.

Most hydrocarbon-based fuels, such as crude oil and natural gas derived from mining operations, are the result of compression and heating of ancient organic materials (i.e., kerogen) over geological time. Formation of petroleum typically occurs from hydrocarbon pyrolysis, in a variety of mostly endothermic reactions at high temperature and/or pressure. Today's oil formed from the preserved remains of prehistoric zooplankton and algae, which had settled to a sea or lake bottom in large quantities under anoxic conditions (the remains of prehistoric terrestrial plants, on the other hand, tended to form coal). Over geological time the organic matter mixed with mud, and was buried under heavy layers of sediment resulting in high levels of heat and pressure (known as diagenesis). This process caused the organic matter to chemically change, first into a waxy material known as kerogen which is found in various oil shales around the world, and then with more heat into liquid and gaseous hydrocarbons in a process known as catagenesis. Most hydrocarbon based fuels derived from crude oil have been undergoing a process of carbon-14 decay over geological time, and, thus, will have little to no detectable carbon-14. In contrast, certain biofuels produced by the living microorganisms of the present invention will comprise carbon-14 at a level comparable to all other presently living things (i.e., an equilibrium level). In this manner, by measuring the carbon-12 to carbon-14 ratio of a hydrocarbon-based biofuel of the present invention, and comparing that ratio to a hydrocarbon based fuel derived from crude oil, the biofuels produced by the methods provided herein can be structurally distinguished from typical sources of hydrocarbon based fuels.

Embodiments of the present invention include methods of producing a commodity chemical, comprising growing a recombinant microorganism with a source of an aldehyde, a ketone, or both, wherein the recombinant microorganism comprises: (i) at least one polynucleotide (i.e., one or more polynucleotides) encoding and expressing a polypeptide having an aldolase activity; and (ii) at least one polynucleotide (i.e., one or more polynucleotides) encoding and expressing a polypeptide having an alcohol dehydrogenase activity, thereby producing the commodity chemical. In certain embodiments, the recombinant microorganism may also comprise at least one polynucleotide (i.e., one or more polynucleotides) encoding and expressing a polypeptide having a double bond reductase activity, and/or at least one polynucleotide encoding and expressing a polypeptide having a dehydratase activity. In certain embodiments, at least one or two of the polynucleotides is an exogenous polynucleotide. In certain embodiments, each of the polynucleotides is exogenous.

Embodiments of the present invention also include recombinant microorganisms that comprise (i) at least one polynucleotide encoding and expressing a polypeptide having an aldolase activity; and (ii) at least one polynucleotide encoding and expressing a polypeptide having an alcohol dehydrogenase activity. In certain embodiments, such microorganisms may also comprise may also comprise at least one polynucleotide encoding and expressing a polypeptide having a double bond reductase activity. In certain embodiments, at least one or two of the polynucleotides is an exogenous polynucleotide. In certain embodiments, each of the polynucleotides is exogenous. These recombinant microorganisms are typically capable of producing a commodity chemical, or an intermediate thereof, from a source of an aldehyde, a ketone, or both.

Embodiments of the present invention also include recombinant microorganisms that comprise (i) at least one polynucleotide encoding and expressing an aldehyde and/or ketone biosynthesis pathway (ii) at least one polynucleotide encoding and expressing a polypeptide having an aldolase activity; and (ii) at least one polynucleotide encoding and expressing a polypeptide having an alcohol dehydrogenase activity. In certain embodiments, such microorganisms may also comprise may also comprise at least one polynucleotide encoding and expressing a polypeptide having a double bond reductase activity. In certain embodiments, at least one or two of the polynucleotides is an exogenous polynucleotide. In certain embodiments, each of the polynucleotides is exogenous. These recombinant microorganisms are typically capable of producing a commodity chemical, or an intermediate thereof, from a source of an aldehyde, a ketone, or both, and may also capable of producing said source of an aldehyde, a ketone, or both, from a suitable monosaccharide or oligosaccharide.

As noted above, in certain embodiments, a recombinant microorganism may also comprise at least one exogenous polynucleotide encoding and expressing a polypeptide having a double bond reductase activity, and/or a polypeptide having a deyhdratase activity. In these and related embodiments, the presence or over-expression of an exogenous double bond reductase or dehydratase may not be necessary to achieve the desired product (e.g., isooctane), as such reactions may be catalyzed by endogenous reductase and dehydratase enzymes, but may be used to increase the amount of desired product produced by the recombinant microorganism.

As used herein, an enzyme or polypeptide having an “aldolase” activity refers generally to a class of enzymes that are capable of catalyzing the reversible reaction of cleaving D-fructose-1,6-bisphosphate to form dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. Aldolase enzymes are present in all animal and plant tissue and in most microorganisms. Class I aldolases, found in animal and higher plant tissue, are typically characterized by not requiring a bivalent metal cofactor and by the formation of a ketimine Schiff base intermediate with the substrate dihydroxyacetone phosphate. Class II aldolases, typically found in microorganisms such as yeast and bacteria, require a metal cofactor and may be inhibited by EDTA.

As one exemplary aldolase, fructose 1,6-biphosphate aldolase (aldolase A) catalyzes a key reaction in glycolysis and energy production. Aldolase B is an isoenzyme of aldolase A, which is capable of cleaving fructose 1-phosphate to form glyceraldehyde and dihydroxyacetone phosphate. This reaction, however, is reversible, and can be utilized to condense glyceraldehyde and dihydroxyacetone phosphate.

Beyond the condensation of glyceraldehye and dihydroxyacetone phosphate, it has been discovered that aldolase enzymes catalyze other useful condensation reactions between aldehydes and/or ketones. In particular, the reversible nature of their catalytic activity has been shown herein to be useful in generating a variety of commodity chemicals, or intermediates thereof, from the condensation products of various combinations of aldehydes and/or ketonees. For instance, an aldolase enzyme of the present invention may be capable of catalyzing the condensation of two molecules of isobutyraldehyde to form 3-hydroxy-2,2,4-trimethyl pentanal. As another example, an aldolase enzyme of the present invention may be capable of catalyzing the condensation of two butyraldehyde molecules to form 3-hydroxy-2-ethylhexanal. As another example, aldolase enzyme of the present invention may be capable of catalyzing the condensation of two hexanaldehyde molecules to form 2-butyl-octanal.

Thus, in certain aspects, the recitation “aldolase” refers to an enzyme that is capable of catalyzing the condensation of various aldehydes and/or ketones, such as acetoaldehyde, propionaldehyde, glutaraldehyde, butyraldehyde, isobutyraldehyde, 2-methyl-butyraldehyde, 3-methyl-butyraldehyde, 4-methylpentaldehyde, hexanaldehyde, heptanaldehyde, octanaldehyde, phenylacetoaldehyde, 2-phenyl acetoaldehyde, 2-(4-hydroxyphenyl)acetaldehyde, 2-Indole-3-acetoaldehyde, 5-amino-pentaldehyde, succinate semialdehyde, and/or succinate 4-hydroxyphenyl acetoaldehyde, among others, including combinations thereof. The condensation of aldehydes and/or ketones may be utilized to produce such chemicals as propanal, butanal, isobutanal, pentanal, 2-methylbutanal, 3-methylbutanal, hexanal, 2-methylpentanal, 3-methylpentanal, 4-methylpentanal, heptanal, octanal, 2-methylheptanal, nonanal, decanal, undecanal, dodecanal, among others recognizable to a person skilled in the art.

“Aldolase” enzymes or “aldolases,” as used herein, also include those enzymes having an acyl-acyl carrier protein (ACP) activity, such as an ACP thioesterase activity. Such ACP esterases typically have the ability to catalyze the production of free fatty acid(s) from fatty acyl-ACP substrates (e.g., C8-C14) under enzyme reactive conditions. However, it is believed that acyl-ACP esterases are also capable of catalyzing the condensation of two aldehydes and/or ketones, such as isobutyeraldehyde and butyeraldehyde, to form a corresponding alkyl aldehyde, such as 3-hydroxy-2,2,4-trimethylpentanal and 2-ethyl-2-hexene-1-al, respectively.

Such esterases are obtainable from the specific exemplified sequences provided herein and from related sources. For example, several species in the genus Cuphea accumulate triglycerides containing medium-chain fatty acids in their seeds, e.g., procumbens, lutea, hookeriana, hyssopifolia, wrightii and inflata. Another natural plant source of medium-chain fatty acids are seeds of the Lauraceae family (e.g., Pisa (Actinodophne hookeri) and Sweet Bay (Laurus nobilis)). Other plant sources include Myristicaceae, Simarubaceae, Vochysiaceae, and Salvadoraceae, and rainforest species of Erisma, Picramnia and Virola, which have been reported to accumulate C14 fatty acids. Exemplary esterases having aldolase activity, including FATB1 and FATB2, are described in U.S. Pat. Nos. 5,298,421, 5,304,481, 5,344,771, 5,455,167, and 5,667,997, and Yuan et al. (PNAS USA 92:10639-634, 1995), which are incorporated by reference for their ACP-acyl thioesterase polynucleotide and polypeptide sequences.

In certain embodiments, a recombinant microorganism may comprise comprise one or more polynucleotide sequences that encode a polypeptide having an aldolase activity, wherein the polynucleotide has 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOS: 215-222, or polynucleotide sequences that hybridize to their complements under conditions described herein and known in the art. SEQ ID NO:215 encodes a FATB2 enzyme from Cuphea hookeriana. SEQ ID NO:216 encodes a FATB3 enzyme from Cuphea lanceolata. SEQ ID NOS:217 and 218 each encode a ketoacyl-ACP synthase IV (KASIV) from Cuphea lanceolata. SEQ ID NO:219 encodes an ACP1-2 enzyme from Cuphea lanceolata, SEQ ID NO:220 encodes an ACP1-3 enzyme from Cuphea lanceolata, and SEQ ID NO:221 encodes an ACP1-1 enzyme from Cuphea lanceolata. SEQ ID NO:222 encodes an Acl1 from Cuphea lanceolata.

Aldolase enzymes may also be obtained from such organisms as Thermotoga maritima and Escherichia coli DH10B, among others. For instance, in certain embodiments, an aldolase enzyme may be based on the polynucleotide reference sequences set forth in SEQ ID NOS:51-82, and/or the polypeptide reference sequences encoded by these polynucleotide reference sequences. Thus, certain recombinant microorganisms of the invention may comprise one or more polynucleotide sequences having 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOS: 51-82, or polynucleotide sequences that hybridize the their complements under conditions of medium or high stringency, as described herein and known in the art.

Certain “aldolase” enzymes of the present invention, or the polynucleotide sequences encoding such enzymes, may also be characterized by certain conserved motifs or domains. In this regard, various amino acid sequences comprising 20 aldolase enzymes from Escerichia coli DH10β were analyzed for common sequence motifs using the PROSITE database. Many of these sequences produced strong matches to known sequence profiles. For instance, ALDOL1, encoded by ECDH10B0008 (SEQ ID NO:63), ALDOL2, encoded by EC ECDH10B0894 (SEQ ID NO:64), ALDOL10, encoded by ECDH10B2629 (SEQ ID NO:72), and ALDOL19, encoded by ECDH10B4135 (SEQ ID NO:81), shared the motifs “Transaldolase signature 1” and “Transaldolase signature 2” (see Table 4, below). Also, ALDOL 5, encoded by ECDH10B1991 (SEQ ID NO:67), contains two motifs, the “KDPG and KHG aldolases active site” motif and the “KDPG and KHG aldolases Schiff-base forming residue” motif (see Table 4, below), and ALDOL6, encoded by ECDH10B2249 (SEQ ID NO:68), ALDOL12, encoded by ECDH10B3100 (SEQ ID NO:74), and ALDOL16, encoded by ECDH10B3310 (SEQ ID NO:78), share the motifs, “Fructose-bisphosphate aldolase class-II signature 1” and “Fructose-bisphosphate aldolase class-II signature 2” (see Table 4, below).

Exemplary aldolase motifs are given below in Table 3, and matching ECDH10B (E. coli DH10B) aldol sequence fragments are given in Table 4.

TABLE 3 Aldolase motifs. Motif Name Sequence Transaldolase  [DGH]-[IVSAC]-T-[ST]- signature 1 N-P-[STA]-[LIVMF] (SEQ ID NO: 223) Transaldolase  [LIVMA]-x-[LIVM]-K- signature 2 [LIVM]-[PAS]-x-[STC]- x-[DENQPAS]-[GC]- [LIVM]-x-[AGV]-x(0,1)-  [QEKRSTH]-x-[LIVMF] (SEQ ID NOS: 224 and 255) KDPG and KHG aldolases G-[LIVM]-x(3)-E-[LIV]-T- active site [LF]-R (SEQ ID NO: 225) KDPG and KHG aldolases G-x(3)-[LIVMF]-K-[LF]-  Schiff-base F-P-[SA]-x(3)-G forming residue (SEQ ID NO: 226) Fructose-bisphosphate [FYVMT]-x(1,3)-[LIVMH]- aldolase class-II [APNT]-[LIVM]-x(1,2)-  signature 1 [LIVM]-H-x-D-H-[GACH] (SEQ ID NOS: 227, 256-260) Fructose-bisphosphate [LIVM]-E-x-E-[LIVM]-G- aldolase class-II x(2)-[GM]-[GSTA]-x-E  signature 2 (SEQ ID NO: 228)

TABLE 4 Motif matches within aldolase sequences. Gene Residues Sequence Match ECDH10B0008 31-39 DATTNPSLI  Transaldolase signature 1 (SEQ ID NO: 229) ECDH10B0008 129-146 ILIKLASTWQGIRAAEQL Transaldolase signature 2 (SEQ ID NO: 230) ECDH10B0894 24-32 GVTTNPSII  Transaldolase signature 1 (SEQ ID NO: 231) ECDH10B0894 82-99 IVVKVPVTAEGLAAIKM Transaldolase signature 2 (SEQ ID NO: 232) ECDH10B2629 30-38 DATTNPSLL  Transaldolase signature 1 (SEQ ID NO: 233) ECDH10B2629 128-145 ILIKLASTWEGIRAAEEL Transaldolase signature 2 (SEQ ID NO: 234) ECDH10B4135 24-32 GVTTNPSII  Transaldolase signature 1 (SEQ ID NO: 235) ECDH10B4135 82-99 IVVKIPVTSEGLAAIKI  Transaldolase signature 2 (SEQ ID NO: 236) ECDH10B1991 40-49 GVRVLEVTLR  KDPG and KHG aldolases (SEQ ID NO: 237) active site ECDH10B1991 128-141 GLKEFKFEPAEANG  KDPG and KHG aldolases (SEQ ID NO: 238) Schiff-base forming residue ECDH10B2249 73-84 YHHPLAIHLDHH  Fructose-bisphosphate aldolase (SEQ ID NO: 239) class-II signature 1 ECDH10B2249 131-142 VEAELGQLGGQE  Fructose-bisphosphate aldolase (SEQ ID NO: 240) class-II signature 2 ECDH10B3100 101-112 YGVPVILHTDHC  Fructose-bisphosphate aldolase (SEQ ID NO: 241) class-II signature 1 ECDH10B3100 172-183 LEIELGCTGGE  Fructose-bisphosphate aldolase (SEQ ID NO: 242) class-II signature 2 ECDH10B3310 71-84 TTYNMPLALHLDHH  Fructose-bisphosphate aldolase (SEQ ID NO: 243) class-II signature 1 ECDH10B3100 131-142 VEAELGRLGGVE  Fructose-bisphosphate aldolase (SEQ ID NO: 244) class-II signature 2

Thus, in certain embodiments, a recombinant microorganism of the invention may comprise one or more polynucleotides that encode an aldolase enzyme or polypeptide, where the aldolase enzyme or polypeptide comprises one or more of the domains or motifs exemplified in Tables 3 and 4, or biologically active variants of these motifs that are capable of facilitating the catalysis of the condensation of various combinations of aldehydes and/or ketones.

An enzyme or polypeptide having an “alcohol dehydrogenase” activity refers generally to an enzyme that catalyzes the conversion of aldehyde or ketone substituents to alcohols or diols, and may include secondary alcohol dehydrogenases. For instance, 2,2,4-trimethylpentanal may be reduced to 2,2,4-trimethyl-1,3-pentanediol by an enzyme having alcohol dehydrogenase (Adh) activity, representing one enzymatic step in the conversion of isobutyraldehyde to isooctane.

In certain aspects, a recombinant microorganism may comprise one or more alcohol dehydrogenases encoded by a nucleotide reference sequence selected from SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 30, 31, 33, and 83-96, or a polypeptide or enzyme encoded by any of these polynucleotide sequences, such as a polypeptide sequence selected from SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, including biologically active fragments or variants thereof, such as optimized variants. Certain recombinant microorganisms of the invention may comprise one or more nucleotide sequences or polypeptide sequences having 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOS:1-34 or 83-96.

For certain of the alcohol dehydrogenase sequences referred to above, SEQ ID NO:1 is the nucleotide sequence and SEQ ID NO:2 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-1: PP_(—)1946) isolated from Pseudomonas putida KT2440. SEQ ID NO:3 is the nucleotide sequence and SEQ ID NO:4 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-2: PP_(—)1817) isolated from Pseudomonas putida KT2440.

SEQ ID NO:5 is the nucleotide sequence and SEQ ID NO:6 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-3: PP_(—)1953) isolated from Pseudomonas putida KT2440. SEQ ID NO:7 is the nucleotide sequence and SEQ ID NO:8 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-4: PP_(—)3037) isolated from Pseudomonas putida KT2440.

SEQ ID NO:9 is the nucleotide sequence and SEQ ID NO:10 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-5: PP_(—)1852) isolated from Pseudomonas putida KT2440. SEQ ID NO:11 is the nucleotide sequence and SEQ ID NO:12 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-6: PP_(—)2723) isolated from Pseudomonas putida KT2440.

SEQ ID NO:13 is the nucleotide sequence and SEQ ID NO:14 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-7: PP_(—)2002) isolated from Pseudomonas putida KT2440. SEQ ID NO:15 is the nucleotide sequence and SEQ ID NO:16 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-8: PP_(—)1914) isolated from Pseudomonas putida KT2440.

SEQ ID NO:17 is the nucleotide sequence and SEQ ID NO:18 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-9: PP_(—)1914) isolated from Pseudomonas putida KT2440. SEQ ID NO:19 is the nucleotide sequence and SEQ ID NO:20 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-10: PP_(—)3926) isolated from Pseudomonas putida KT2440.

SEQ ID NO:21 is the nucleotide sequence and SEQ ID NO:22 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-11: PFL_(—)1756) isolated from Pseudomonas fluorescens Pf-5. SEQ ID NO:23 is the nucleotide sequence and SEQ ID NO:24 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-12: KPN_(—)01694) isolated from Klebsiella pneumoniae subsp. pneumoniae MGH 78578.

SEQ ID NO:25 is the nucleotide sequence and SEQ ID NO:26 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-13: KPN_(—)02061) isolated from Klebsiella pneumoniae subsp. pneumoniae MGH 78578. SEQ ID NO:27 is the nucleotide sequence and SEQ ID NO:28 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-14: KPN_(—)00827) isolated from Klebsiella pneumoniae subsp. pneumoniae MGH 78578.

SEQ ID NO:29 is the nucleotide sequence and SEQ ID NO:30 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-16: KPN_(—)01350) isolated from Klebsiella pneumoniae subsp. pneumoniae MGH 78578. SEQ ID NO:31 is the nucleotide sequence and SEQ ID NO:32 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-17: KPN_(—)03369) isolated from Klebsiella pneumoniae subsp. pneumoniae MGH 78578. SEQ ID NO:33 is the nucleotide sequence and SEQ ID NO:34 is the polypeptide sequence of a secondary alcohol dehydrogenase (2adh-18: KPN_(—)03363) isolated from Klebsiella pneumoniae subsp. pneumoniae MGH 78578.

The alcohol dehydrogenase encoding polynucleotide sequences of SEQ ID NOS: 83-96 were obtained from Pseudomonas putida.

In certain aspects, an alcohol dehydrogenase (adh), a secondary alcohol dehydrogenase (2adh), a fragment, variant, or derivative thereof, or any other enzyme that utilizes such an active site, may comprise at least one of a nicotinamide adenine dinucleotide (NAD+), NADH, nicotinamide adenine dinucleotide phosphate (NADP+), or NADPH binding motif. In certain embodiments, the NAD+, NADH, NADP+, or NADPH binding motif may be selected from the group consisting of Y-X-G-G-X-Y (SEQ ID NO:245), Y-X-X-G-G-X-Y (SEQ ID NO:246), Y-X-X-X-G-G-X-Y (SEQ ID NO:247), Y-X-G-X-X-Y (SEQ ID NO:248), Y-X-X-G-G-X-X-Y (SEQ ID NO:249), Y-X-X-X-G-X-X-Y (SEQ ID NO:250), Y-X-G-X-Y (SEQ ID NO:251), Y-X-X-G-X-Y (SEQ ID NO:252), Y-X-X-X-G-X-Y (SEQ ID NO:253), and Y-X-X-X-X-G-X-Y (SEQ ID NO:254); wherein Y is independently selected from alanine, glycine, and serine, wherein G is glycine, and wherein X is independently selected from a genetically encoded amino acid.

In certain embodiments, a microbial system or recombinant microorganism may comprise natural or optimized alcohol dehydrogenases from Pseudomonads, Rhodococcus erythropolis ATCC4277, Norcadia fusca AKU2123, Klebsialla, or other suitable organisms. Genes encoding alcohol dehydrogenases may be isolated from these and other organisms according to known techniques in the art and incorporated into the recombinant microorganisms as described herein.

An enzyme or polypeptide having a “double bond reductase” activity refers generally to an enzyme that catalyzes the following general reaction:

As noted herein, in certain embodiments, the above reaction may be catalyzed by the endogenous enzymes in a given microorganism, such that the addition of an exogenous enzyme having double bond reductase activity may not be necessary to practice the present invention. Nonetheless, it is believed that in certain aspects the over-expression of a double bond reductase enzyme, or the expression of a particular double bond reductase having increased affinity for the components of the specific reaction contemplated, may allow increased production of the desired commodity chemical, or intermediate thereof.

Double bond reductases may be obtained, for example, from such organisms as Saccharomyces cerevisiae, Pichia angusta, Zymomonas mobilis, E. coli, Klebsiella pneumoniae, Pseudomonas fluorescens, and Pseudomonas putida, among others.

In certain embodiments, a double bond reductase enzyme may be based on the polynucleotide reference sequences set forth in SEQ ID NOS:35-50, and/or the corresponding polypeptide reference sequences encoded by these polynucleotides. Thus, certain recombinant microorganisms of the invention may comprise one or more polynucleotide sequences having 80%, 85%, 90%, 95%, 97%, 98%, 99% sequence identity to SEQ ID NOS: 35-50, or polynucleotide sequences that hybridize the their complements under conditions described herein and known in the art.

The “double bond reductase” enzymes of the present invention, or the polynucleotide sequences encoding such enzymes, may also be characterized by certain conserved motifs or domains, as described herein and known in the art.

In certain embodiments, the source of the aldehyde, the ketone, or both, which is condensed by an enzyme having aldolase activity to form a commodity chemical, or intermediate thereof, may include a recombinant microorganism that comprises an aldehyde and/or ketone biosynthesis pathway. In certain embodiments, such a biosynthesis pathway may include an aldehyde biosynthesis pathway, a ketone biosynthesis pathway, or both.

In certain embodiments, the biosynthesis pathway may include one or more of an acetoaldehyde, propionaldehyde, glutaraldehyde, butyraldehyde, isobutyraldehyde, 2-methyl-butyraldehyde, 3-methyl-butyraldehyde, 4-methylpentaldehyde, phenylacetoaldehyde, 2-phenyl acetoaldehyde, 2-(4-hydroxyphenyl)acetoaldehyde, 2-Indole-3-acetoaldehyde, 5-amino-pentaldehyde, succinate semialdehyde, and/or succinate 4-hydroxyphenyl acetaldehyde biosynthesis pathway, including various combinations thereof.

In certain aspects, the biosynthesis pathway comprises a butyraldehyde or isobutyraldehyde biosynthesis pathway. Exemplary aldehyde and ketone biosynthesis pathways are described herein and in U.S. application Ser. Nos. 12/245,537 and 12/245,540, which are incorporated by reference for their description of aldehyde/ketone biosynthesis pathways.

In certain aspects, a propionaldehyde biosynthesis pathway may comprise a threonine deaminase (ilvA) gene from an organism such as Escherichia coli and a keto-isovalerate decarboxylase (kivd) gene from an organism such as Lactococcus lactis, and/or functional variants of these enzymes, including homologs or orthologs thereof, as well as optimized variants. These enzymes may be utilized generally to convert L-threonine to propionaldehyde.

In certain aspects, a butyraldehyde biosynthesis pathway may comprise at least one of a thiolase (atoB) gene from an organism such as E. coli, a β-hydroxy butyryl-CoA dehydrogenase (hbd) gene, a crotonase (crt) gene, a butyryl-CoA dehydrogenase (bcd) gene, an electron transfer flavoprotein A (etfA) gene, and/or an electron transfer flavoprotein B (etfB) gene from an organism such as Clostridium acetobutyricum (e.g., ATCC 824), as well as a coenzyme A-linked butyraldehyde dehydrogenase (ald) gene from an organism such as Clostridium beijerinckii acetobutyricum ATCC 824. In certain aspects, a coenzyme A-linked alcohol dehydrogenase (adhE2) gene from an organism such as Clostridium acetobutyricum ATCC 824 may be used as an alternative to an ald gene.

In certain aspects, an isobutyraldehyde biosynthetic pathway may comprise an acetolactate synthase (alsS) from an organism such as Bacillus subtilis or an als gene from an organism such as Klebsiella pneumoniae subsp. pneumoniae MGH 78578 (codon usage may be optimized for E. coli protein expression). Such a pathway may also comprise acetolactate reductoisomerase (ilvC) and/or 2,3-dihydroxyisovalerate dehydratase (ilvD) genes from an organism such as E. coli, as well as a keto-isovalerate decarboxylase (kivd) gene from an organism such as Lactococcus lactis.

In certain aspects, a 3-methylbutyraldehyde and 2-methylbutyraldehyde biosynthesis pathway may comprise an acetolactate synthase (alsS) gene from an organism such as Bacillus subtilis or an (als) gene from an organism such as Klebsiella pneumoniae subsp. pneumoniae MGH 78578 (codon usage may be optimized for E. coli protein expression). Certain aspects of such a pathway may also comprise acetolactate reductoisomerase (ilvC), 2,3-dihydroxyisovalerate dehydratase (ilvD), isopropylmalate synthase (LeuA), isopropylmalate isomerase (LeuC and LeuD), and 3-isopropylmalate dehydrogenase (LeuB) genes from an organism such as E. coli, as well as a keto-isovalerate decarboxylase (kivd) from an organism such as Lactococcus lactis.

In certain aspects, a phenylacetoaldehyde and 4-hydroxyphenylacetoaldehyde biosynthesis pathway may comprise one or more of 3-deoxy-7-phosphoheptulonate synthase (aroF, aroG, and aroH), 3-dehydroquinate synthase (aroB), a 3-dehydroquinate dehydratase (aroD), dehydroshikimate reductase (aroE), shikimate kinase II (aroL), shikimate kinase I (aroK), 5-enolpyruvylshikimate-3-phosphate synthetase (aroA), chorismate synthase (aroC), fused chorismate mutase P/prephenate dehydratase (pheA), and/or fused chorismate mutase T/prephenate dehydrogenase (tyrA) genes from an organism such as E. coli, as well as a keto-isovalerate decarboxylase (kivd) from an organism such as Lactococcus lactis.

In certain embodiments, the recombinant microrganism that is the source of the aldehyde, the ketone, or both, may be the same recombinant microorganism that converts the aldehyde or ketone to the commodity chemical. For example, in the production of isooctane, such a recombinant microorganism may comprise an isobutyraldehyde biosynthesis pathway, an aldolase enzyme, optionally a double bond reductase enzyme, and an alcohol dehydrogenase enzyme, and would be capable of converting a suitable monosaccharide to isobutyraldehyde, and then to 3-hydroxy-2,2,4-trimethyl pentanal and 2,2,4-trimethyl-1,3-pentanediol. In these and related aspects, 2,2,4-trimethyl-1,3-pentanediol may be further converted to isooctane, such as by “hydrotreating,” as described herein and known in the art.

As a further example, in the production of commodity chemicals such as 2-ethylhexanol and 2-ethylhexane, such a recombinant microorganism may comprise a butyraldehyde biosynthesis pathway, an aldolase enzyme, optionally a double bond reductase enzyme, and an alcohol dehydrogenase enzyme, and would be capable of converting a suitable monosacharide to butyraldehyde, and then to 3-hydroxy-2-ethyl hexanal, 2-ethyl-2-hexene-1-al, 2-ethylhexanal, and finally to 2-ethylhexanol. In these and related aspects, 2-ethylhexanol may be further converted to 2-ethylhexane, such as by “hydrotreating,” as described herein and known in the art.

In certain embodiments, the recombinant microorganism that is the source of the aldehyde, the ketone, or both may be different than the recombinant microorganism that converts the aldehyde or ketone to the commodity chemical. In these and related embodiments, a first recombinant microorganism that comprises an aldehyde and/or ketone biosynthesis pathway may be utilized as a source or feedstock to produce one or more aldehydes and or ketones, which may then be converted to the desired commodity chemical, such as 2,2,4-trimethyl-1,3-pentanediol or 2-ethylhexanol, by a second recombinant microorganism that comprises an aldolase, optionally an exogenous double bond reductase, and an alchohol dehydrogenase. In these and related embodiments, the two recombinant microorganisms may be cultured together. Alternatively, the aldehyde, ketone, or both may be produced separately by the first recombinant microorganism and then later incubated with the second recombinant microorganism that comprises the aldolase and the alcohol dehydrogenase. Various other combinations and aspects of the invention will be apparent to a person skilled in the art in this regard.

In other embodiments, the source of an aldehyde and/or ketone, such as isobutyraldehyde, may be any other suitable source, such as a commercially available source. In these and any embodiments, the “source” of the aldehyde, ketone, or both may include the aldehyde or the ketone itself, which may be added directly to a microbial culture system as needed.

As for all other pathways and enzymes described herein, the aldolases, optional double bond reductases, and alcohol dehydrogenases, and the components for each of the aldehyde and/or ketone biosynthesis pathways described herein may be present in a recombinant microorganism either endogenously or exogenously. To improve the efficiency of a given biosynthesis pathway, endogenous genes, for example, may be up-regulated or over-expressed, such as by introducing an additional copy ((i.e., exogenous gene) of that otherwise endogenous gene into the recombinant microorganism. Such pathways may also be optimized by altering via mutagenesis the endogenous version of a gene to improve functionality, followed by introduction of the altered gene into the microorganism. The expression of endogenous genes may be up or down-regulated, or even eliminated, according to known techniques in the art and described herein. Similarly, the expression levels of exogenous genes or polynucleotide sequences may be regulated as desired, such as by using various constitutive or inducible promoters. Such genes or polynucleotides may also be “codon-optimized,” as described herein and known in the art. Also included are functional naturally-occurring variants of the genes and enzymes/polypeptides described herein, including homologs or orthologs thereof.

Any microorganism may be utilized according to the present invention. In certain aspects, a microorganism is a eukaryotic or prokaryotic microorganism. In certain aspects, a microrganism is a yeast, such as S. cerevisiae. In certain aspects, a microorganism is a bacteria, such as a gram-positive bacteria or a gram-negative bacteria. Given its rapid growth rate, well-understood genetics, the variety of available genetic tools, and its capability in producing heterologous proteins, genetically modified E. coli may be used in certain embodiments of a microbial system as described herein, whether for the degradation and metabolism of a polysaccharide, such as alginate or pectin, or the formation or biosynthesis of commodity chemicals, such as biofuels.

Other microorganisms may be used according to the present invention, based in part on the compatibility of enzymes and metabolites to host organisms. For example, other organisms such as Acetobacter aceti, Achromobacter, Acidiphilium, Acinetobacter, Actinomadura, Actinoplanes, Aeropyrum pernix, Agrobacterium, Alcaligenes, Ananas comosus (M), Arthrobacter, Aspargillus niger, Aspargillus oryze, Aspergillus melleus, Aspergillus pulverulentus, Aspergillus saitoi, Aspergillus sojea, Aspergillus usamii, Bacillus alcalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus lentus, Bacillus licheniformis, Bacillus macerans, Bacillus stearothermophilus, Bacillus subtilis, Bifidobacterium, Brevibacillus brevis, Burkholderia cepacia, Candida cylindracea, Candida rugosa, Carica papaya (L), Cellulosimicrobium, Cephalosporium, Chaetomium erraticum, Chaetomium gracile, Clostridium, Clostridium butyricum, Clostridium acetobutylicum, Clostridium thermocellum, Corynebacterium (glutamicum), Corynebacterium efficiens, Escherichia coli, Enterococcus, Erwina chrysanthemi, Gliconobacter, Gluconacetobacter, Haloarcula, Humicola insolens, Humicola nsolens, Kitasatospora setae, Klebsiella, Klebsiella oxytoca, Kluyveromyces, Kluyveromyces fragilis, Kluyveromyces lactis, Kocuria, Lactlactis, Lactobacillus, Lactobacillus fermentum, Lactobacillus sake, Lactococcus, Lactococcus lactis, Leuconostoc, Methylocystis, Methanolobus siciliae, Methanogenium organophilum, Methanobacterium bryantii, Microbacterium imperiale, Micrococcus lysodeikticus, Microlunatus, Mucor javanicus, Mycobacterium, Myrothecium, Nitrobacter, Nitrosomonas, Nocardia, Papaya carica, Pediococcus, Pediococcus halophilus, Penicillium, Penicillium camemberti, Penicillium citrinum, Penicillium emersonii, Penicillium roqueforti, Penicillum lilactinum, Penicillum multicolor, Paracoccus pantotrophus, Propionibacterium, Pseudomonas, Pseudomonas fluorescens, Pseudomonas denitrificans, Pyrococcus, Pyrococcus furiosus, Pyrococcus horikoshii, Rhizobium, Rhizomucor miehei, Rhizomucor pusillus Lindt, Rhizopus, Rhizopus delemar, Rhizopus japonicus, Rhizopus niveus, Rhizopus oryzae, Rhizopus oligosporus, Rhodococcus, Sccharomyces cerevisiae, Sclerotina libertina, Sphingobacterium multivorum, Sphingobium, Sphingomonas, Streptococcus, Streptococcus thermophilus Y-1, Streptomyces, Streptomyces griseus, Streptomyces lividans, Streptomyces murinus, Streptomyces rubiginosus, Streptomyces violaceoruber, Streptoverticillium mobaraense, Tetragenococcus, Thermus, Thiosphaera pantotropha, Trametes, Trichoderma, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Trichosporon penicillatum, Vibrio alginolyticus, Xanthomonas, yeast, Zygosaccharomyces rouxii, Zymomonas, and Zymomonus mobilis, may be utilized as recombinant microorganisms provided herein, and, thus, may be utilized according to the various methods of the present invention.

The various embodiments described herein can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

The following Examples are offered by way of illustration, not limitation.

EXAMPLES Example 1 Aldehyde/Ketone Biosynthesis Pathways

To provide a useful source of aldehydes and/or ketones, such as butyraldehyde and isobutyraldehyde, recombinant microorganisms that comprise an aldehyde and/or ketone biosynthesis pathway, and which are capable of converting a suitable monosaccharide or oligosaccharide to an aldehyde or ketone, were constructed.

A butyraldehyde biosynthetic pathway comprising a thiolase (atoB) gene from E. coli, β-hydroxy butyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd), electron transfer flavoprotein A (etfA), and electron transfer flavoprotein B (etfB) genes from Clostridium acetobutyricum ATCC 824, and a coenzyme A-linked butyraldehyde dehydrogenase (ald) gene from Clostridium beijerinckii acetobutyricum ATCC 824 was constructed in E. coli and tested for the ability to produce butyraldehyde. Also, a coenzyme A-linked alcohol dehydrogenase (adhE2) gene from Clostridium acetobutyricum ATCC 824 was used as an alternative to ald and tested for the ability to produce butanol.

An isobutyraldehyde biosynthetic pathway comprising an acetolactate synthase (alsS) from Bacillus subtilis or (als) from Klebsiella pneumoniae subsp. pneumoniae MGH 78578 (codon usage was optimized for E. coli protein expression) and acetolactate reductoisomerase (ilvC) and 2,3-dihydroxyisovalerate dehydratase (ilvD), genes from E. coli and keto-isovalerate decarboxylase (kivd) from Lactococcus lactis was constructed and tested for the ability to produce isobutyraldehyde, as measured by isobutanal production.

Construction of pBADButP.

The DNA sequence encoding hbd, crt, bcd, etfA, and etfA of Clostridium acetobutyricum ATCC 824 was amplified by polymerase chain reaction (PCR) from 50 ng Clostridium acetobutyricum ATCC 824 genome (ATCC) in 50 μl. Amplified DNA fragment was digested with BamHI and XbaI and ligated into pBAD33 pre-digested with the same restriction enzymes.

Construction of pBADButP-atoB.

The DNA sequence encoding atoB of Escherichia coli DH10B was amplified by polymerase chain reaction (PCR) from 50 ng Escherichia coli DH10B genome in 50 μl. Amplified DNA fragment was digested with XbaI and PstI and ligated into pBADButP pre-digested with the same restriction enzymes.

Construction of pBADatoB-ald.

The DNA sequence encoding atoB of Escherichia coli DH10B and ald from Clostridium beijerinckii were amplified separately by polymerase chain reaction (PCR) from either 50 ng Escherichia coli DH10B or Clostridium beijerinckii genome (ATCC) in 50 μl, respectively. The amplified DNA fragments were gel purified and eluted into 30 ul of EB buffer (Qiagen). 5 ul from each DNA solution was combined and each DNA fragment was spliced by another round of PCR. The spliced fragment was digested with SacI and HindIII and ligated into pBADButP pre-digested with the same restriction enzymes.

Construction of pBADButP-atoB-ALD.

The DNA fragment 1 encoding chloramphenicol acetyltransferase (CAT), P15 origin of replication, araBAD promoter, atoB of Escherichia coli DH10B and ald of Clostridium beijerinckii and the DNA fragment 2 encoding araBAD promoter, hbd, crt, bcd, etfA, and etfB of Clostridium acetobutyricum ATCC 824 were amplified separately by polymerase chain reaction (PCR) from 50 ng of either pBADatoB-ald or pBADButP in 50 μl, respectively. Amplified DNA fragments were digested with NotI and KpnI and ligated each other.

Construction of pBADals-ilvCD.

The DNA fragment encoding als of Klebsiella pneumoniae subsp. pneumoniae MGH 78578 of its codon usage optimized for over-expression in E. coli was amplified by polymerase chain reaction (PCR) from 50 ng pETals in 50 μl. The amplified DNA fragment was digested with SacI and XbaI and ligated into pBADilvCD pre-digested with the same restriction enzymes.

Construction of pBADalsS-ilvCD.

The DNA fragments encoding front and bottom halves of alsS of Bacillus subtilis B26 were amplified by polymerase chain reaction (PCR) from 50 ng Bacillus subtilis B26 genome (ATCC) in 50 μl. The amplified DNA fragments were gel purified and eluted into 30 ul of EB buffer (Qiagen). 5 ul from each DNA solution was combined and each DNA fragment was spliced by another round of PCR. The spliced fragment was internal XbaI site free and thus was digested with SacI and XbaI and ligated into pBADilvCD pre-digested with the same restriction enzymes.

Construction of pTrcBALK.

A DNA sequence encoding ketoisovalerate decarboxylase (kivd) of Lactococcus lavtis was amplified by polymerase chain reaction (PCR) 50 ng pETBAL in 50 μl. Amplified DNA fragment was digested with SacI and XbaI and ligated into pTrcBAL pre-digested with the same restriction enzymes.

Construction of pTrcBALD.

A DNA sequence encoding CoA-linked aldehyde dehydrogenase (ald) of Clostridium beijerinckii was amplified by polymerase chain reaction (PCR) from 50 ng pETBAL in 50 μl. Amplified DNA fragment was digested with SacI and HndIII and ligated into pTrcBAL pre-digested with the same restriction enzymes.

Construction of pBBRPduCDEGH.

A DNA sequence encoding propanediol dehydratase medium (pduD) and small (pduE) subunits and propanediol dehydratase reactivation large (pduG) and small (pduH) subunits of Klebsiella pneumoniae subsp. pneumoniae MGH 78578 was amplified by polymerase chain reaction (PCR) from 50 ng Klebsiella pneumoniae subsp. pneumoniae MGH 78578 in 50 μl. Amplified DNA fragment was digested with SacII and XbaI and ligated into pTrc99A pre-digested with the same restriction enzymes to form pBBRPduDEGH.

A DNA sequence encoding propanediol dehydratase large subunit (pduC) of Klebsiella pneumoniae subsp. pneumoniae MGH 78578 was amplified by polymerase chain reaction (PCR) from 50 ng Klebsiella pneumoniae subsp. pneumoniae MGH 78578 in 50 μl. Amplified DNA fragment was digested with XhoI and XbaI and ligated into pBBRPduDEGH pre-digested with the same restriction enzymes.

To test the butyraldehyde biosynthesis pathway, DH10B harboring pBADButP-atoB/pTrcBALD and pBADButP-atoB-ALD/pTrcB2DH/pBBRpduCDEGH were grown overnight in LB media containing 50 ug/ml chroramphenicol (Cm⁵⁰) and 100 ug/ml ampicillin (Amp¹⁰⁰) at 37 C, 200 rpm. An aliquot of each seed culture was inoculated into fresh TB media containing Cm⁵⁰ and Amp¹⁰⁰ and was grown in incubation shaker at 37 C, 200 rpm. Three hours after inoculation, the cultures were induced with 13.3 mM arabinose and 1 mM IPTG and were grown for overnight. 700 ul of this culture was extracted with equal volume of ethylacetate and analyzed by GC-MS.

To test the isobutyeraldehyde biosynthesis pathway, DH10B cells harboring pBADals-ilvCD/pTrcBALK or pBADalsS-ilvCD/pTrcBALK were grown overnight in LB media containing 50 ug/ml chroramphenicol (Cm⁵⁰) and 100 ug/ml ampicillin (Amp¹⁰⁰) at 37 C, 200 rpm. An aliquot of each seed culture was inoculated into fresh TB media containing Cm⁵⁰ and Amp¹⁰⁰ and was grown in an incubation shaker at 37° C., 200 rpm. Three hours after inoculation, the cultures were induced with 13.3 mM arabinose and 1 mM IPTG and were grown for overnight. 700 ul of this culture was extracted with equal volume of ethylacetate and analyzed by GC-MS for the production of isobutyraldehyde, as measured by isobutanal production. FIG. 2 shows the production of isobutanal from these cultures. This strain also produced 2-methylbutyraldehyde and 3-methylbutyraldehyde as minor components (not shown).

Example 2 Production of 3-Hydroxy-2,2,4 Trimethylpentanal from Isobutyraldehyde

The ability of an enzyme having aldolase activity to condense two molecules of isobutyraldehyde to form 3-hydroxy-2,2,4-trimethylpentanal was tested in vivo. Plasmid TrcTM1559 was constructed as described in Example 5 below, and contains an aldose-encoding polynucleotide sequence that was obtained from Thermotoga maritima.

A single colony of E. coli DH10B stain harboring pTrcTM1559 was inoculated into fresh LB media containing Amp¹⁰⁰ and the culture was grown in an incubation shaker at 37° C. for overnight. One percent of this culture was inoculated into fresh TB media containing Amp¹⁰⁰ and the culture was grown in incubation shaker at 37° C. When the culture was grown to an OD_(600nm) of 0.6, the culture was induced with 0.5 mM IPTG. 50 mM isobutyraldehyde was added and the cultures were grown for a day. 500 ul of culture was extracted with 500 ul of ethylacetate, and the extracts were analyzed by GC-MS. The formation of 3-hydroxy-2,2,4-trimethylpentanal was observed (see FIG. 2).

Example 3 Production of 2,2,4-Trimethyl-1,3-Pentanediol from Isobutyraldehyde

The ability of a recombinant microorganism comprising an aldolase enzymen and an alcohol dehydrogenase to convert isobutyraldehyde to 2,2,4-trimethyl-1,3-pentanediol is tested. Plasmid TrcTM1559 is described in Example 2 above. Various alcohol dehydrogenases were isolated from Pseudomonas putida KT2440, Pseudomonas fluorescens Pf-5, and Klebsiella pneumoniae MGH 78578 (see SEQ ID NO:1-34) and cloned into expression plasmids, as described in U.S. application Ser. Nos. 12/245,537 and 12/245,540, which are incorporated by reference for their description, construction and testing of alcohol dehydrogenase enzymes. Additional alcohol dehydrogenases were also obtained from Pseudomonas putida (see SEQ ID NOS:83-96).

A single colony of E. coli DH10B stain harboring both pTrcTM1559 and an alcohol dehydrogenase expressing plasmid is inoculated into fresh LB media containing Amp¹⁰⁰ and the culture is grown in incubation shaker at 37° C. for overnight. One percent of this culture is inoculated into fresh TB media containing Amp¹⁰⁰ and the culture is grown in incubation shaker at 37° C. When the culture is grown to an OD_(600nm) of 0.6, the culture is induced with 0.5 mM IPTG. 50 mM isobutyraldehyde is added and the cultures are grown for a day. 500 ul of culture is extracted with 500 ul of ethylacetate, and the extracts are analyzed by GC-MS. The formation of 2,2,4-trimethylpentandiol is observed.

Example 4 Production of Other Medium to Long Chain Hydrocarbons

In addition to abovementioned pathway to produce 2,2,4-trimethyl-1,3-pentanediol and then 2,2,4, trimethylpentane (isooctane), enzymatic aldol condensation followed by alcohol dehydrogenation can yield variety of medium to long chain hydrocarbons from various aldehydes and ketones as starting materials. These aldehydes and/or ketones can be produced by recombinant microorganisms according to the aldehyde and/or ketone biosynthesis pathways described in U.S. application Ser. Nos. 12/245,537 and 12/245,540, which are incorporated by reference for the description, construction, and testing of these pathways.

One example is illustrated herein, in which two molecules of butyraldehyde were condensed by an aldolase to form 3-hydroxy-2-ethyl hexanal. This molecule was then spontaneously or enzymatically dehydrated to form 2-ethyl-2-hexene-1-al, which may then be consecutively reduced to form 2-ethylhexanal and 2-ethylhexanol, catalyzed by double bond reductase and alcohol dehydrogenase, respectively.

Also illustrated herein is a reaction in which two molecules of hexanaldehyde were condensed by an aldolase to form 3-hydroxy-2-butyl-1-octanal. This molecule was then spontaneously or enzymatically dehydrated to form 2-butyl-2-octene-1-al, which may then be consecutively reduced to for 2-butyl-octanal and 2-butyl octanol.

To test the production of 2-ethyl-2-hexene-1-al, a single colony of E. coli DH10B stain harboring pTrcTM1559 was inoculated into fresh LB media containing Amp¹⁰⁰ and the culture grown in incubation shaker at 37 C for overnight. One percent of this culture was inoculated into fresh TB media containing Amp¹⁰⁰ and the culture was grown in incubation shaker at 37 C. When the culture was grown OD_(600nm) of 0.6, the culture was induced with 0.5 mM IPTG. Either 50 mM butyraldehyde or 50 mm hexanaldehyde was added and the cultures were grown for a day. 500 ul of the culture was extracted with 500 ul of ethylacetate, and the extracts were analyzed by GC-MS. The formation of 2-ethyl-2-hexene-1-al was observed (see FIG. 3). Also, the formation of 2-butyl-2-octene-1-al was observed (see FIG. 4).

Example 5 Isolation and Cloning of Aldolases

Polynucleotide sequences that encode enzymes having aldolase activity were isolated from Thermotoga maritima and Escherichia coli DH10B and cloned into expression vectors.

-   -   Construction of pTrcTM0040, pTrcTM0066, pTrcTM0273, pTrcTM0283,         pTrcTM0295, pTrcTM0343, pTrcTM0720, pTrcTM1072, pTrcTM1419,         pTrcTM1521, pTrcTM1559, and pTrcTM1744

As the DNA sequences encoding TM0273, TM0283, TM0720, TM 1559, and TM1744 contain an internal NcoI site, flanking regions of NcoI site for each gene were amplified separately by polymerase chain reaction (PCR): 98° C. for 10 sec, 60° C. for 15 sec, and 72° C. for 30 min, repeated 30 times. The reaction mixture contained 1× Phusion buffer (NEB), 2 mM dNTP, 0.5 μM forward and reverse primers (see Table 5), 1U Phusion High Fidelity DNA polymerase (NEB), and 50 ng Thermotoga maritima genome in 50 μl.

TABLE 5 Gene name Forward 5′ -> 3′ Reverse 5′ -> 3′ TM0273  CATGCCATGGGG CTGCGGTACCATAGAAGCTCCGTGA fragment 1 ATGACAATGCCTTATGTAAAGAACA (SEQ ID NO: 98) (SEQ ID NO: 97) TM0273 fragment 2

GCTCTAGA TTACGCCTTTCCGCTGGATCCGAAT (SEQ ID NO: 100) TM0283  CATGCCATGGGG TGCCCACACCATCGCGAATGTTGAA fragment 1 ATGAGGAGTACAGACAGGTTACTGT (SEQ ID NO: 102) (SEQ ID NO: 101) TM0283 fragment 2

GCTCTAGA TTACTTCTGACCGTACTTGGTGTGG (SEQ ID NO: 104) TM0720  CATGCCATGGGG CATGCTTCCCATTGTTTCTATGACA fragment 1 ATGTGGAAGCATGTAAAACAGGTTG (SEQ ID NO: 106) (SEQ ID NO: 105) TM0720 fragment 2

CACCGGTGCTCCGTGGGTGAGATGT (SEQ ID NO: 108) TM0720 fragment 3

GCTCTAGA TTATATTTCCACCCCTTCGATCTTG (SEQ ID NO: 110) TM1559  CATGCCATGGGG CACACAGACTCCGTGGAAACGATTT fragment 1 ATGATAGAGTACAGGATTGAGGAGG (SEQ ID NO: 112) (SEQ ID NO: 111) TM1559 fragement 2

GCTCTAGA TTAACCTCCATATCTCTCTTCTCCC (SEQ ID NO: 114) TM1744  CATGCCATGGGG AGCCTGTGCCATCGCTTTTCTCATC fragment 1 ATGATCGATCTCAGGTCCGACACCG (SEQ ID NO: 116) (SEQ ID NO: 115) TM1744 fragment 2

TTGATTTCCCATTGTGCCGGAGGGT (SEQ ID NO: 118) TM1744 fragment 3

GAGAACCGCCATCGCTCCGACCTCG (SEQ ID NO: 120) TM1744 fragment 4

ATCGGGGTCCATCGCTCCATTTTTT (SEQ ID NO: 122) TM1744 fragment 5

GCTCTAGA TTAGGAGAATTTTCTGAAGAGTTTT (SEQ ID NO: 124)

The DNA sequences encoding TM0040, TM0066, TM0273, TM0283, TM0295, TM0343, TM0720, TM1072, TM1419, TM1521, TM1559, and TM1744 of Themotoga maritima were amplified by polymerase chain reaction (PCR): 98° C. for 10 sec, 60° C. for 15 sec, and 72° C. for 1 min, repeated 30 times. The reaction mixture contained 1× Phusion buffer (NEB), 2 mM dNTP, 0.5 μM forward and reverse primers (see Table 6), 1U Phusion High Fidelity DNA polymerase (NEB), and 50 ng Thermotoga maritima genome in 50 μl (Note: For TM0273, TM0283, TM0720, TM 1559, and TM1744, fragments prepared as described in the preceding paragraph were used). The amplified DNA fragments were digested with NcoI and XbaI and ligated into pTrc99A pre-digested with the same restriction enzymes.

TABLE 6 Gene name Forward 5′ -> 3′ Reverse 5′ -> 3′ TM0044 CATGCC GCTCTAGA  ATGGTTTACACAACTCCCTGGAACAGAAAG  TTACAACCTTTGCCAGAGTATCGCC (SEQ ID NO: 125) (SEQ ID NO: 126) TM0066 CATGCCATGGGG GCTCTAGA  ATGAAGATGGAAGAGCTCTTCAAAA  TTATTCTGTGCACCCCCTGATCTTT (SEQ ID NO: 127) (SEQ ID NO: 128) TM0273 CATGCCATGGGG GCTCTAGA  ATGACAATGCCTTATGTAAAGAACA  TTACGCCTTTCCGCTGGATCCGAAT (SEQ ID NO: 129) (SEQ ID NO: 130) TM0283 CATGCCATGGGG GCTCTAGA  ATGAGGAGTACAGACAGGTTACTGT  TTACTTCTGACCGTACTTGGTGTGG (SEQ ID NO: 131) (SEQ ID NO: 132) TM0295 CATGCCATGGGG GCTCTAGA  ATGAAGATCTTTCTGGACACAGCAA  TTATTTCTTCAGGTTCTCCAAATAC (SEQ ID NO: 133) (SEQ ID NO: 134) TM0343 CATGCCATGGGG GCTCTAGA ATGATAGTCGTTTTGAAACCCGGTT  TTAATTCACCTTCACCCCCAGGGCA (SEQ ID NO: 135) (SEQ ID NO: 136) TM0720 CATGCCATGGGG GCTCTAGA  ATGTGGAAGCATGTAAAACAGGTTG  TTATATTTCCACCCCTTCGATCTTG (SEQ ID NO: 137) (SEQ ID NO: 138) TM1072 CATGCCATGGGG GCTCTAGA  ATGAGAGAGACGATAAGAGAGATTC  TTACAGCCATCCCTCCGGAACACCC (SEQ ID NO: 139) (SEQ ID NO: 140) TM1419 CATGCC   GCTCTAGA  ATGGTCAAGGTCCTGATCCTCGGTC TTACAGCCACTTCGGTTTCAATCCC (SEQ ID NO: 141) (SEQ ID NO: 142) TM1521 CATGCCATGGGG GCTCTAGA  ATGTTCAGAGGAGTAGGAACTGCTA  TTATAGCAATCCACTCTCCTTGAGA (SEQ ID NO: 143) (SEQ ID NO: 144) TM1559 CATGCCATGGGG GCTCTAGA  ATGATAGAGTACAGGATTGAGGAGG  TTAACCTCCATATCTCTCTTCTCCC (SEQ ID NO: 145) (SEQ ID NO: 146) TM1744 CATGCCATGGGG GCTCTAGA  ATGATCGATCTCAGGTCCGACACCG  TTAGGAGAATTTTCTGAAGAGTTTT (SEQ ID NO: 147) (SEQ ID NO: 148)

Construction of pTrcDH10Bxxx

The DNA sequences encoding EC1648 and EC4071 contain an internal NcoI site, and the DNA sequence encoding EC2249 contained an XbaI site. Flanking regions of the restriction sites for each gene were amplified separately by polymerase chain reaction (PCR): 98° C. for 30 sec, 55° C. for 15 sec, and 72° C. for 45 sec, repeated 30 times. The reaction mixture contained 1× Phusion buffer (NEB), 2 mM dNTP, 0.3 μM forward and reverse primers (see Table 7), 1U Phusion High Fidelity DNA polymerase (NEB), and 20 ng E. coli DH10β.

TABLE 7 Gene name Forward 5′ -> 3′ Reverse 5′ -> 3′ EC1648 CATGCCATGGGGATGGCAGATTTAGACGATATTAAAG CTGAAAATAACCGTGGTCAAAAGCC fragment 1 (SEQ ID NO: 149) (SEQ ID NO: 150) EC1648 fragment 2

CAGTCACGGCCATCGTCGGCATTCCC (SEQ ID NO: 152) EC1648 fragment 3

GCTCTAGATTACTGTTTTTCACTCAGATAG (SEQ ID NO: 154) EC2249 CATGCCATGGGGATGTACGTGGTATCGACAAAG GTTCTCCAGTCTCGAAAAATCAAGC fragment 1 (SEQ ID NO: 155) (SEQ ID NO: 156) EC2249 fragment 2

GCTCTAGATTATGCCCTGCCCTCGCAGC (SEQ ID NO: 158) EC4071 CATGCCATGGGGATGAATAAGTACACCATCAAC GAGGATCACCCACGGCATATTGATATG fragment 1 (SEQ ID NO: 159) (SEQ ID NO: 160) EC4071 fragment 2

GCTCTAGATTAGCGGCGTTTAGCCATC (SEQ ID NO: 162)

The DNA sequences encoding EC0008, EC0894, EC0940, EC1648, EC1991, EC2249, EC2250, EC2465, EC2629, EC2969, EC3100, EC3233, EC3299, EC3305, EC3310, EC4071, EC4092, EC4135 and EC4539 of Escherichia coli DH10β were amplified by polymerase chain reaction (PCR): 98° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 1 min, repeated 30 times. The reaction mixture contained 1× Phusion buffer (NEB), 2 mM dNTP, 0.3 μM forward and reverse primers (see Table 8), 1U Phusion High Fidelity DNA polymerase (NEB), and 20 ng Escherichia coli DH10β genome in 50 μl (Note: For EC1648, EC2249 and EC4071 fragments prepared in the preceding paragraph were used).

TABLE 8 Gene name Forward 5′ -> 3′ Reverse 5′ -> 3′ EC0008 CATGCCATGGGGATGA  GCTCTAGATTACAGCA  CGGACAAATTGACCTC GATCGCCGATC (SEQ ID NO: 163) (SEQ ID NO: 164) EC0894 CATGCCATGGGGATGG GCTCTAGATTAAATCG AACTGTATCTGGATAC  ACGTTCTGCCAAAC (SEQ ID NO: 165) (SEQ ID NO: 166) EC0940 CATGCCATGGGGATGA GCTCTAGATTAACGCG  TTGATTTACGCAGTGA CCAGGAATGCAC TAC (SEQ ID NO: 168) (SEQ ID NO: 167) EC1648 CATGCCATGGGGATGG GCTCTAGATTACTGTT CAGATTTAGACGATAT TTTCACTCAGATAG TAAAG (SEQ ID NO: 170) (SEQ ID NO: 169) EC1991 CATGCCATGGGGATGA GCTCTAGATTACAGCT  AAAACTGGAAAACAAG  TAGCGCCTTCTAC (SEQ ID NO: 171) (SEQ ID NO: 172) EC2249 CATGCCATGGGGATGT GCTCTAGATTATGCCC ACGTGGTATCGACAAA TGCCCTCGCAGC  G (SEQ ID NO: 174) (SEQ ID NO: 173) EC2250 CATGCCATGGGGATGA GCTCTAGATCAGGCGA  CAGATATTGCGCAGTT TAGTAATTTTGC G (SEQ ID NO: 176) (SEQ ID NO: 175) EC2465 CATGCCATGGGGATGG GCTCTAGATTAACGCT  CACAACCTGCCGCTAT GCCAGCTTAAGG TATTC (SEQ ID NO: 178) (SEQ ID NO: 177) EC2629 CATGCCATGGGGATGA GCTCTAGATTATAGTT ACGAGTTAGACGGCAT TGGCGGCAAGAAG C (SEQ ID NO: 180) (SEQ ID NO: 179) EC2969 CATGCCATGGGGATGG  GCTCTAGATTACTCTT  AACGAAATAAACTTGC CAATTCGTAACC (SEQ ID NO: 181) (SEQ ID NO: 182) EC3100 CATGCCATGGGGATGT GCTCTAGATTACAGAA CTAAGATTTTTGATTT CGTCGATCGCGTTC C (SEQ ID NO: 184) (SEQ ID NO: 183) EC3233 CATGCCATGGGGATGG  GCTCTAGATTAATTAT ATATTGTATTTATAGA TTTCTTTCAGATTATT G G (SEQ ID NO: 185) (SEQ ID NO: 186) EC3299 CATGCCATGGGGATGA GCTCTAGATTATTTTT ATAACGATGTTTTCCC  TAAAGGTATCAG  (SEQ ID NO: 187) (SEQ ID NO: 188) EC3305 CATGCCATGGGGATGA GCTCATATATATATCC AACATCTGACAGAAAT TCCTTTATTGGCCTTC G ACAGGCTGTG (SEQ ID NO: 189) (SEQ ID NO: 190) EC3310 CAATAAAGGAGGATAT GCTCTAGATTATGCTG  ATATATGAGCATTATC AAATTCGATTCG TCCACTAAATATC (SEQ ID NO: 192) SEQ ID NO: 191) EC4071 CATGCCATGGGGATG  GCTCTAGATTAGCGGC AATAAGTACACCATC GTTTAGCCATC  AAC (SEQ ID NO: 194) (SEQ ID NO: 193) EC4092 CATGCCATGGGGATGC GCTCTAGATTACAGCG AAAACATTACTCAGTC  CCAGCGCACTGG  (SEQ ID NO: 195) (SEQ ID NO: 196) EC4135 CATGCCATGGGGATGG  GCTCTAGATTAGAGAT AACTGTATCTGGACAC GAGTAGTGCCAAATG (SEQ ID NO: 197) (SEQ ID NO: 198) EC4539 CATGCCATGGGGATGA  GCTCTAGATTAGTAGC CTGATCTGAAAGCAAG TGCTGGCGCTC (SEQ ID NO: 199) (SEQ ID NO: 200)

The amplified fragments encoding EC3305 and EC3310 were ligated together using the overlap-PCR method. (PCR): 98° C. for 30 sec, 55° C. for 30 sec, and 72° C. for 1 min, repeated 30 times. The reaction mixture contained 1× Phusion buffer (NEB), 2 mM dNTP, 0.3 μM forward and reverse primers (forward primer: catgccatggggatgaaacatctgacagaaatg (SEQ ID NO:189), reverse primer: gctctagattatgctgaaattcgattcg (SEQ ID NO:190)), 1U Phusion High Fidelity DNA polymerase (NEB), and 3 μL PCR product from the previous paragraph.

Amplified DNA fragment was digested with NcoI and XbaI and ligated into pTrc99A pre-digested with the same restriction enzymes. 

1. A recombinant microorganism, comprising: a) hexanal; b) 2-butyl-2-octene-1-al and c) recombinant DNA comprising SEQ ID NO:51 wherein said recombinant DNA encodes an aldolase wherein said aldolase is expressed in said microorganism and said hexanal is converted to 2-butyl-2-octene-1-al by said aldolase.
 2. The microorganism of claim 1 wherein the microorganism is yeast.
 3. The microorganism of claim 1 wherein the microorganism is E. coli. 